Treatment of amyotrophic lateral sclerosis

ABSTRACT

The present invention relates to the identification of compounds and pharmaceutical compositions thereof for treating subjects with amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. The invention also provides methods of preparing the provided compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 61/116,571, filed Nov. 20, 2008, the entirety of which is herebyincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R43NS057849 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerativedisorder caused by motor neuron death (Rowland et al., N. Engl. J. Med.,2001, 334, 1688-1700) and characterized in part by the presence ofabnormal aggregates of insoluble protein in selectively vulnerablepopulations of neurons and glia. ALS, an orphan disease, is estimated toafflict about 87,000 people worldwide, but its prevalence would be muchhigher were it not for the fact that ALS patients survive for only 3 to5 years on average after diagnosis. Approximately 10% of ALS cases arefamilial, with the rest of the cases being sporadic (Rowland et al., N.Engl. J. Med., 2001, 334, 1688-1700). Approximately 20% of the cases offamilial ALS are caused by inherited mutations in the protein Cu/Znsuperoxide dismutase (SOD1) (Rosen et al., Nature, 1993, 362, 59-62).Rodent models in mutant SOD1 are often used as a disease model becauseof its phenotypic and pathologic resemblance to sporadic and familialhuman ALS (Dal Canto et al., Brain Res., 1995, 676, 25-40; Wong, et al.,Neuron, 1995, 14, 1105-1116; Bruijin et al., Science, 1998, 281,1851-1854; Bruijn et al., Neuron, 1997, 18, 327-338; Wang et al., Hum.Mol. Genet., 2003, 12, 2753-2764; Wang et al., Neurobiol. Dis., 2002,10, 128-138; Jonsson et al., Brain, 2004, 127, 73-88).

The causes of sporadic ALS remain unknown, and the clinical courses arevariable, suggesting that multiple factors are involved. Differenthypotheses have been proposed, such as glutamate-mediatedexcitotoxicity, impaired mitochondrial function, oxidative stress, andaberrant protein aggregation (Dib et al., Drugs, 2003, 63, 289-310;Strong et al., Pharmacology & Therapeutics, 2003, 98, 379-414; Kunst etal., Am. J. Hum. Genet., 2004, 75, 933-947; Bruijn et al., Annu. Rev.Neurosci, 2004, 27, 723-749; Dibernardo et al., Biochimica et BiophysicaActa, 2006, 1762, 1139-1149). Riluzole, which decreases glutamateexcitotoxicity, is the only FDA approved ALS drug (Jimonet et al., J.Med. Chem., 1999, 42, 2828-2843). However, it can only extend mediansurvival life for 2 to 3 months, suggesting mechanisms other thanglutamate-mediated excitotoxicity should be considered during ALS drugdevelopment (Miller et al., ALS and Other Motor Neuron Disorders, 2003,4, 191-206; Taylor et al., Neurology, 2006, 67, 20-27).

Imbalances in protein homeostasis, which can be caused by cell stress orexpression of certain mutant proteins, can result in the appearance ofalternative conformational states that are able to self-associate toform protein aggregates and inclusion bodies. In familial as well assporadic forms of ALS and mutant SOD1 transgenic models, aberrantprotein aggregation has been reported as a common feature (Bruijin etal., Science, 1998, 281, 1851-1854; Leigh et al., Cytoskeletalpathologyin Motor Neuron Diseases, in: Rowland, L. P. (Editor), AmyotrophicLateral Sclerosis and Other Motor Neuron Disease, Raven Press, New York,1991, pp. 3-pp. 23; Mather et al., Neurosci. Lett., 1993, 160, 13-16;Pasinelli et al., Neuron, 2004, 43, 19-30; Watanabe, et al., Neruobiol.Dis., 2001, 8, 933-941). The ubiquity of this feature in various formsof ALS suggests that perhaps drugs that inhibit aberrant proteinaggretation may provide new and improved treatment options. Thus, thereis an urgent need to identify and develop drugs for the treatment ofALS.

SUMMARY OF THE INVENTION

The present invention encompasses the recognition that there exists aneed new compounds and methods for treating patients with amyotrophiclateral sclerosis (ALS) or other neurodegenerative diseasescharacterized by the presence of aberrant protein aggregates.

The present invention relates to the identification of providedcompounds and pharmaceutical compositions thereof to treatneurodegenerative diseases. Among other things, the present inventionprovides methods of treating amyotrophic lateral sclerosis (ALS) withprovided compounds. Without wishing to be bound by any particulartheory, provided compounds may be useful in the treatment of ALS orother neurodegenerative diseases where abnormal protein aggregation hasbeen implicated, as they may prevent the aggregation of protein in acell or limit the toxicity of such aggregates.

In one aspect, the invention provides compounds of the formula:

or a pharmaceutically acceptable salt thereof, or a tautomer thereof,wherein R⁰, R¹, R², R³, n, and p are as defined herein. In someembodiments, such compounds, or pharmaceutically acceptable saltsthereof, or tautomers thereof, are used in the treatment ofneurodegenerative diseases.

In another aspect, the invention provides methods for treating a subjectwith amyotrophic lateral sclerosis (ALS) or other neurodegenerativedisease (e.g., Alzheimer's Disease, prion disease, or Huntington'sDisease) by administering a therapeutically effective amount of aninventive compound, or a pharmaceutical composition thereof. In certainembodiments, the compound is of one of the classes described herein oris a species described herein. In another aspect, the invention providesmethods for treating a subject with ALS or other neurodegenerativedisease by administering both an inventive compound or pharmaceuticalcomposition thereof, and a second therapeutic agent or pharmaceuticalcomposition thereof. The two compounds and/or compositions may beadministered as a combination composition comprising both compounds.Alternatively, the two compounds may be administered separately (e.g.,as two different compositions) either simultaneously or sequentially.

The present invention also provides methods of identifying compoundsthat protect against the cytotoxic effects of abnormal proteinaggregation. Additionally, the present invention provides methods ofidentifying compounds that prevent, inhibit, or reverse proteinaggregation. In some embodiments, the inventive assay involves the useof PC12 cells expressing the protein SOD1. In certain embodiments, SOD1is labeled with a detectable moiety (e.g., a fluorescent moiety). Assaysmay be high-throughput assays.

In another aspect, the present invention provides methods of inhibitingor reversing abnormal protein aggregation (e.g., SOD1 proteinaggregates). Inhibiting or reversing abnormal protein aggregation mayoccur in vivo (e.g., in a subject as described herein) or in vitro(e.g., in a cell). In yet another aspect, the invention provides methodsof protecting cells from the cytotoxic effects of aggregated protein(e.g., SOD1) using an inventive compound. Protection of cells may occurin vivo or in vitro. In some embodiments, protection occurs in vitro andthe cells are PC12 cells. In another aspect, the invention providesmethods of modulating proteasome activity in vivo or in vitro using aprovided compound. In some embodiments, protection occurs in vitro, andthe cells used are PC12 cells or HeLa cells. In certain embodiments, thecells are mammalian cells. In certain embodiments, the cells are in anorganism (e.g., a mammal).

The present invention contemplates provided compounds for use inmedicine.

All publications and patent documents cited in this application areincorporated herein by reference in their entirety.

DEFINITIONS

Aliphatic: The term “aliphatic” or “aliphatic group,” as used herein,means a straight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-12aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-6 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-5 aliphatic carbon atoms. In other embodiments, aliphaticgroups contain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic C₃-C₆ hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic, that has a single point of attachment to the rest of themolecule. Suitable aliphatic groups include, but are not limited to,linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynylgroups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkyl: As used herein, the term “alkyl” refers to saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups.

Alkylene: The term “alkylene” refers to a bivalent alkyl group. An“alkylene chain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein nis a positive integer, preferably from 1 to 20, 1 to 12, 1 to 6, from 1to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylenechain is a polymethylene group in which one or more methylene hydrogenatoms are replaced with a substituent. Suitable substituents includethose described below for a substituted aliphatic group.

Alkenyl: As used herein, the term “alkenyl” refers to unsaturatedaliphatic groups analogous in possible substitution to the alkylsdescribed above, but that contain at least one double bond.

Alkenylene: As used herein, the term “alkenylene” refers to a bivalentalkenyl group. A substituted alkenylene chain is a polymethylene groupcontaining at least one double bond in which one or more hydrogen atomsare replaced with a substituent. Suitable substituents include thosedescribed below for a substituted aliphatic group.

Alkynyl: As used herein, the term “alkynyl” refers to unsaturatedaliphatic groups analogous in possible substitution to the alkylsdescribed above, but that contain at least one triple bond.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, a genetically-engineered animal,and/or a clone.

Approximately: As used herein, the terms “approximately” or “about” inreference to a number are generally taken to include numbers that fallwithin a range of 5%, 10%, 15%, or 20% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evidentfrom the context (except where such number would be less than 0% orexceed 100% of a possible value). In some embodiments, use of the term“about” in reference to dosages means±5 mg/kg/day.

Aryl: The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl,” as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

Arylalkyl: As used herein, the term “arylalkyl” refers to an arylsubstituted alkyl group, wherein the terms “aryl” and “alkyl” aredefined herein, and wherein the aryl group is attached to the alkylgroup, which in turn is attached to the parent molecule. An exemplaryarylalkyl group includes benzyl.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, 10, 15, or more amino acids are required tobe characteristic of a protein. In general, a characteristic portion isone that, in addition to the sequence identity specified above, sharesat least one functional characteristic with the relevant intact protein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Effective amount: As used herein, an “effective amount” is an amountthat achieves, or is expected to achieve, a desired result, and can beadministered in one dose or in multiple doses. Compositions may beconsidered to contain an effective amount if they include a dose that iseffective in the context of a dosing regimen, even if the composition asa single dose is not expected to be effective.

Heteroaryl: The term “heteroaryl,” used alone or as part of a largermoiety as in “heteroaralkyl” or “heteroarylalkoxy,” refers tomonocyclic, bicyclic, and tricyclic ring systems having a total of fiveto fourteen ring members, wherein one or more ring in the system isaromatic, one or more ring in the system contains one or moreheteroatoms, and wherein each ring in the system contains 3 to 7 ringmembers. The term “heteroatom” refers to nitrogen, oxygen, or sulfur,and includes any oxidized form of nitrogen or sulfur, and anyquaternized form of a basic nitrogen. Heteroaryl groups include, withoutlimitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl,triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl,isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. Theterms “heteroaryl” and “heteroar-,” as used herein, also include groupsin which a heteroaromatic ring is fused to one or more aryl,cycloaliphatic, or heterocyclyl rings, where the radical or point ofattachment is on the heteroaromatic ring. Nonlimiting examples includeindolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, andpyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono- orbicyclic. The term “heteroaryl” may be used interchangeably with theterms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any ofwhich terms include rings that are optionally substituted. The term“heteroaralkyl” refers to an alkyl group substituted by a heteroaryl,wherein the alkyl and heteroaryl portions independently are optionallysubstituted.

Heteroatom: The term “heteroatom” means one or more of oxygen, sulfur,nitrogen, phosphorus, or silicon (including, any oxidized form ofnitrogen, sulfur, phosphorus, or silicon; the quaternized form of anybasic nitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,”“heterocyclic radical,” and “heterocyclic ring” are used interchangeablyand refer to a stable 5- to 7-membered monocyclic or 7-10-memberedbicyclic heterocyclic moiety that is either saturated or partiallyunsaturated, and having, in addition to carbon atoms, one or more,preferably one to four, heteroatoms, as defined above. When used inreference to a ring atom of a heterocycle, the term “nitrogen” includesa substituted nitrogen. As an example, in a saturated or partiallyunsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Intraperitoneal: The phrases “intraperitoneal administration” and“administered intraperitonealy” as used herein have their art-understoodmeaning referring to administration of a compound or composition intothe peritoneum of a subject.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.,animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, and/or microbe).

Lower alkyl: The term “lower alkyl” refers to a C₁₋₄ straight orbranched alkyl group. Exemplary lower alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term “lower haloalkyl” refers to a C₁₋₆ straight orbranched alkyl group that is substituted with one or more halogen atoms.

Optionally substituted: As described herein, compounds of the inventionmay contain “optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Unless otherwise indicated, suitable monovalent substituents on asubstitutable carbon atom of an “optionally substituted” group areindependently halogen; —(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O(CH₂)₀₋₄R^(o),—O—(CH₂)₀₋₄C(O)OR^(o); —(CH₂)₀₋₄—CH(OR^(o))₂; —(CH₂)₀₋₄SR^(o);—(CH₂)₀₋₄Ph, which may be substituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Phwhich may be substituted with R^(o); —CH═CHPh, which may be substitutedwith R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted withR^(o); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R^(o))C(O)R^(o);—N(R^(o))C(S)R^(o); —(CH₂)₀₋₄N(R^(o))C(O)NR^(o) ₂; —N(R^(o))C(S)NR^(o)₂; —(CH₂)₀₋₄N(R^(o))C(O)OR^(o); —N(R^(o))N(R^(o))C(O)R^(o);—N(R^(o))N(R^(o))C(O)NR^(o) ₂; —N(R^(o))N(R^(o))C(O)OR^(o);—(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄OC(O)R^(o);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(o); —(CH₂)_(o-4)SC(O)R^(o);—(CH₂)₀₋₄C(O)NR^(o) ₂; —C(S)NR^(o) ₂; —C(S)SR^(o); —SC(S)SR^(o),—(CH₂)₀₋₄OC(O)NR^(o) ₂; —C(O)N(OR^(o))R^(o); —C(O)C(O)R^(o);—C(O)CH₂C(O)R^(o); —C(NOR^(o))R^(o); —(CH₂)₀₋₄SSR^(o);—(CH₂)₀₋₄S(O)₂R^(o); —(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄OS(O)₂R^(o);—S(O)₂NR^(o) ₂; —(CH₂)₀₋₄S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂;—N(R^(o))S(O)₂R^(o); —N(OR^(o))R^(o); —C(NH)NR^(o) ₂; —P(O)₂R^(o);—P(O)R^(o) ₂; —OP(O)R^(o) ₂; —OP(O)(OR^(o))₂; SiR^(o) ₃; —(C₁₋₄ straightor branched)alkylene)O—N(R^(o))₂; or —(C₁₋₄ straight or branched)alkylene)C(O)O—N(R^(o))₂, wherein each R^(o) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(o), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Unless otherwise indicated, suitable monovalent substituents on R^(o)(or the ring formed by taking two independent occurrences of R^(o)together with their intervening atoms), are independently halogen,—(CH₂)₀₋₂R., -(haloR.), —(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR., —(CH₂)₀₋₂CH(OR.)₂;—O(haloR.), —CN, —N₃, —(CH₂)₀₋₂C(O)R., —(CH₂)₀₋₂C(O)OH,—(CH₂)₀₋₂C(O)OR., —(CH₂)₀₋₂SR., —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂,—(CH₂)₀₋₂NHR., —(CH₂)₀₋₂NR.₂, —NO₂, —SiR.₃, —OSiR.₃, —C(O)SR., —(C₁₋₄straight or branched alkylene)C(O)OR., or —SSR. wherein each R. isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(o) include ═O and ═S.

Unless otherwise indicated, suitable divalent substituents on asaturated carbon atom of an “optionally substituted” group include thefollowing: ═O, ═S, ═NNR*₂, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*,═NOR*, —O(C(R*₂))₂₋₃O—, or —S(C(R*₂))₂₋₃S—, wherein each independentoccurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may besubstituted as defined below, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Unlessotherwise indicated, suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Unless otherwise indicated, suitable substituents on the aliphatic groupof R* include halogen, —R., -(haloR.), —OH, —OR., —O(haloR.), —CN,—C(O)OH, —C(O)OR., —NH₂, —NHR., —NR.₂, or —NO₂, wherein each R. isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Unless otherwise indicated, suitable substituents on a substitutablenitrogen of an “optionally substituted” group include —R^(†), —NR^(†) ₂,—C(O)R^(†), —C(O)OR^(†), —C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†),—S(O)₂NR^(†) ₂, —C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†);wherein each R^(†) is independently hydrogen, C₁₋₆ aliphatic which maybe substituted as defined below, unsubstituted —OPh, or an unsubstituted5-6-membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Unless otherwise indicated, suitable substituents on the aliphatic groupof R^(†) are independently halogen, —R., -(haloR.), —OH, —OR.,—O(haloR.), —CN, —C(O)OH, —C(O)OR., —NH₂, —NHR., —NR.₂, or —NO₂, whereineach R. is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Oral: The phrases “oral administration” and “administered orally” asused herein have their art-understood meaning referring toadministration by mouth of a compound or composition.

Parenteral: The phrases “parenteral administration” and “administeredparenterally” as used herein have their art-understood meaning referringto modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal, and intrasternal injection and infusion.

Partially unsaturated: As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Patient: As used herein, the term “patient,” “subject,” or “testsubject” refers to any organism to which a provided compound isadministered in accordance with the present invention e.g., forexperimental, diagnostic, prophylactic, and/or therapeutic purposes.Typical subjects include animals (e.g., mammals such as mice, rats,rabbits, non-human primates, and humans; insects; worms; etc.). In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition (e.g., a neurodegenerative disease,a disease, disorder or condition associated with protein aggregation,ALS, etc.).

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Prodrug: A general, a “prodrug,” as that term is used herein and as isunderstood in the art, is an entity that, when administered to anorganism, is metabolized in the body to deliver a therapeutic agent ofinterest. Various forms of “prodrugs” are known in the art. For examplesof such prodrug derivatives, see:

-   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and    Methods in Enzymology, 42:309-396, edited by K. Widder, et al.    (Academic Press, 1985);-   b) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen;-   c) Bundgaard, Chapter 5 “Design and Application of Prodrugs,” by H.    Bundgaard, p. 113-191 (1991);-   d) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);-   e) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285    (1988); and-   f) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

The methods and structures described herein relating to the providedcompounds also apply to pharmaceutically acceptable salts thereof.

Protecting group: The term “protecting group,” as used herein, is wellknown in the art and include those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd)edition, John Wiley & Sons, 1999, the entirety of which is incorporatedherein by reference. Suitable amino-protecting groups include methylcarbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromo ethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Unless otherwise indicated, suitably protected carboxylic acids furtherinclude, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, andarylalkyl-protected carboxylic acids. Examples of suitable silyl groupsinclude trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples ofsuitable alkyl groups include methyl, benzyl, p-methoxybenzyl,3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples ofsuitable alkenyl groups include allyl. Examples of suitable aryl groupsinclude optionally substituted phenyl, biphenyl, or naphthyl. Examplesof suitable arylalkyl groups include optionally substituted benzyl(e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.

Unless otherwise indicated, suitable hydroxyl protecting groups includemethyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoro ethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, a-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,a-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). In some embodimments, proteins include onlynaturally-occurring amino acids. In some embodiments, proteins includeone or more non-naturally-occurring amino acids (e.g., moieties thatform one or more peptide bonds with adjacent amino acids). In someembodiments, one or more residues in a protein chain contains anon-amino-acid moiety (e.g., a glycan, etc). In some embodiments, aprotein includes more than one polypeptide chain, for example linked byone or more disulfide bonds or associated by other means. In someembodiments, proteins contain L-amino acids, D-amino acids, or both; insome embodiments, proteins contain one or more amino acid modificationsor analogs known in the art. Useful modifications include, e.g.,terminal acetylation, amidation, methylation, etc. The term “peptide” isgenerally used to refer to a polypeptide having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids. In some embodiments, proteinsare antibodies, antibody fragments, biologically active portionsthereof, and/or characteristic portions thereof.

Provided compound: The term “provided compound,” as used herein, refersto a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:p is 1-2;

designates a single or double bond, wherein one

is a single bond and one

is a double bond;

-   each R¹ is independently —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R,    —N(R)₂, —CN, —NO₂—NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR,    —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R,    —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or an optionally substituted 3-8    membered saturated, partially unsaturated, or aryl monocyclic ring    having 0-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or an optionally substituted 8-10 membered saturated,    partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; or    -   two R¹ groups are taken together to form

-   -    wherein:        -   X is N or C;        -   each R⁵ is independently —R, —OR, —SR, —S(O)R, —SO₂R,            —OSO₂R, —N(R)₂, —CN, —NO₂—NRC(O)R, —NRC(O)(CO)R,            —NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR,            —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂,            or a 3-8 membered saturated, partially unsaturated, or aryl            monocyclic ring having 0-4 heteroatoms independently            selected from nitrogen, oxygen, or sulfur, or an 8-10            membered saturated, partially unsaturated, or aryl bicyclic            ring having 0-4 heteroatoms independently selected from            nitrogen, oxygen, or sulfur, wherein R⁵ is optionally            substituted with 1-5 R groups;

-   each R is independently hydrogen, halogen, optionally substituted    C₁₋₂₀ aliphatic, optionally substituted C₁₋₂₀ heteroaliphatic,    optionally substituted phenyl, optionally substituted arylalkyl, or    two R on the same nitrogen are taken together to form a 5-6 membered    saturated, partially saturated, or aryl ring having 1-3 heteroatoms    independently selected from nitrogen, oxygen, and sulfur, or two R    on the same carbon or on adjacent carbons are optionally taken    together to form a 3-6 membered saturated cycloalkyl or fused    monocyclic ring containing 0-2 heteroatoms independently selected    from nitrogen, oxygen, and sulfur;

-   n is 0-1;

-   each R² and R³ are independently —R, —OR, —SR, —S(O)R, —SO₂R,    —OSO₂R, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂,    a suitable amino protecting group, or an optionally substituted 3-8    membered saturated, partially unsaturated, or aryl monocyclic ring    having 0-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or an optionally substituted 8-10 membered saturated,    partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;

-   R⁰ is R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, N(R)₂, —NRC(O)R,    —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R,    —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂,    —OC(O)N(R)₂, or

-    wherein:    -   L is a valence bond or a bivalent saturated or partially        unsaturated C₁₋₁₀ hydrocarbon chain, wherein 1-4 methylene units        of L are optionally and independently replaced by —O—, —S—,        —C(O)—, —OC(O)—, —C(O)O—, —OC(O)O—, —S(O)—, —S(O)₂—, —OSO₂O—,        —N(R)C(O)—, —C(O)NR—, —N(R)C(O)O—, —OC(O)NR—, —N(R)C(O)NR—,        —N(R⁶)—, or -Cy-, and wherein L is optionally substituted with        1-4 R groups; wherein:        -   each -Cy- is independently a bivalent optionally substituted            saturated, partially unsaturated, or aromatic monocyclic or            bicyclic ring selected from a 6-10 membered arylene, a 5-10            membered heteroarylene having 1-4 heteroatoms independently            selected from oxygen, nitrogen, or sulfur, a 3-8 membered            carbocyclylene, or a 3-10 membered heterocyclylene having            1-4 heteroatoms independently selected from oxygen,            nitrogen, or sulfur;        -   R⁶ is —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —C(O)R, —C(O)OR,            —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, a suitable amino            protecting group, or an optionally substituted 3-8 membered            saturated, partially unsaturated, or aryl monocyclic ring            having 0-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, or an optionally substituted 8-10            membered saturated, partially unsaturated, or aryl bicyclic            ring having 0-4 heteroatoms independently selected from            nitrogen, oxygen, or sulfur;    -   Ring A is a 3-8 membered saturated, partially unsaturated, or        aryl monocyclic ring optionally containing 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, or an        8-10 membered saturated, partially unsaturated, or aryl bicyclic        ring optionally containing 0-4 heteroatoms independently        selected from nitrogen, oxygen, or sulfur, and wherein Ring A is        optionally substituted with occurrences of R⁴;        -   m is 0-5; and    -   each R⁴ is independently —R, —OR, —SR, —CN, —S(O)R, —SO₂R,        —OSO₂R, —N(R)₂, —NO₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂,        —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR,        —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or a 3-8 membered        saturated, partially unsaturated, or aryl monocyclic ring        optionally containing 0-4 heteroatoms independently selected        from nitrogen, oxygen, or sulfur.

In some embodiments, the term “provided compound” may encompass prodrugsand/or esters of compounds of formula I. As discussed herein, providedcompounds may be provided in salt form. In particular, in someembodiments, a provided compound is provided as a pharmaceuticallyacceptable salt of a compound of formula I.

Provided compounds may exist in particular geometric or stereoisomericforms. The present invention contemplates all such compounds, includingcis- and trans-isomers, R- and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, the racemic mixtures thereof, and othermixtures thereof, as falling within the scope of the invention.Additional asymmetric carbon atoms may be present in a substituent suchas an alkyl group. All such isomers, as well as mixtures thereof, areintended to be included in this invention. That is, in some embodiments,the present invention provides isolated single isomers. In someembodiments, the present invention provides mixtures of two or moreisomers. In certain embodiments, the present invention relates to aprovided compound represented by any of the structures outlined herein,wherein the compound is provided as a single stereoisomer.

The terms acid or base addition salt also comprise the hydrates and thesolvent addition forms which the compounds are able to form. Examples ofsuch forms are, e.g., hydrates, alcoholates and the like.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants, which are in themselves known, but are not mentioned here.

Stereochemically isomeric forms: The phrase “stereochemically isomericforms,” as used herein, refers to different compounds made up of thesame atoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable. In someembodiments of the invention, chemical compositions may be provided aspure preparations of individual stereochemically isomeric forms of acompound; in some embodiments, chemical compositions may be providedthat are or include mixtures of two or more stereochemically isomericforms of the compound. In certain embodiments, such mixtures containequal amounts of different stereochemically isomeric forms; in certainembodiments, such mixtures contain different amounts of at least twodifferent stereochemically isomeric forms. In some embodiments, achemical composition may contain all diastereomers and/or enantiomers ofthe compound. In some embodiments, a chemical composition may containless than all diastereomers and/or enantiomers of a compound. Unlessotherwise indicated, the present invention encompasses allstereochemically isomeric forms of relevant compounds, whether in pureform or in admixture with one another. If a particular enantiomer of acompound of the present invention is desired, it may be prepared, forexample, by asymmetric synthesis, or by derivation with a chiralauxiliary, where the resulting diastereomeric mixture is separated andthe auxiliary group cleaved to provide the pure desired enantiomers.Alternatively, where the molecule contains a basic functional group,such as amino, diastereomeric salts are formed with an appropriateoptically-active acid, and resolved, for example, by fractionalcrystallization.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and/or chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with and/or displays oneor more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Tautomeric forms: The phrase “tautomeric forms,” as used herein, is usedto describe different isomeric forms of organic compounds that arecapable of facile interconversion. Tautomers may be characterized by theformal migration of a hydrogen atom or proton, accompanied by a switchof a single bond and adjacent double bond. In some embodiments,tautomers may result from prototropic tautomerism (i.e., the relocationof a proton). In some embodiments, tautomers may result from valencetautomerism (i.e., the rapid reorganization of bonding electrons). Allsuch tautomeric forms are intended to be included within the scope ofthe present invention. In some embodiments, tautomeric forms of acompound exist in mobile equilibrium with each other, so that attemptsto prepare the separate substances results in the formation of amixture. In some embodiments, tautomeric forms of a compound areseparable and isolatable compounds. In some embodiments of theinvention, chemical compositions may be provided that are or includepure preparations of a single tautomeric form of a compound. In someembodiments of the invention, chemical compositions may be provided asmixtures of two or more tautomeric forms of a compound. In certainembodiments, such mixtures contain equal amounts of different tautomericforms; in certain embodiments, such mixtures contain different amountsof at least two different tautomeric forms of a compound. In someembodiments of the invention, chemical compositions may contain alltautomeric forms of a compound. In some embodiments of the invention,chemical compositions may contain less than all tautomeric forms of acompound. In some embodiments of the invention, chemical compositionsmay contain one or more tautomeric forms of a compound in amounts thatvary over time as a result of interconversion. Unless otherwiseindicated, the present invention encompasses all tautomeric forms ofrelevant compounds, whether in pure form or in admixture with oneanother.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount. In some embodiments, a composition maybe considered to include a therapeutically effective amount of aprovided compound when it includes an amount that is effective whenadministered as part of a dosing regimen even if a single dose (i.e.,only the amount in the composition) alone is not expected to beeffective.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, delay onset of, reduce severity of, and/orreduce incidence of one or more symptoms or features of a disease,disorder, and/or condition; in some embodiments, treatment prevents oneor more symptoms of features of the disease, disorder, or condition.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition. In some embodiments, treatmentmay be administered to a subject who exhibits only early signs of thedisease, disorder, and/or condition, for example for the purpose ofdecreasing the risk of developing pathology associated with the disease,disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administeredsystemically,” “peripheral administration,” and “administeredperipherally” as used herein have their art-understood meaning referringto administration of a compound or composition such that it enters therecipient's system.

Unsaturated: The term “unsaturated,” as used herein, means that a moietyhas one or more units of unsaturation.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The methods and structures described herein relating to compounds andcompositions of the invention also apply to the pharmaceuticallyacceptable acid or base addition salts and all stereoisomeric forms ofthese compounds and compositions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Proteasome inhibition is selectively toxic to PC12 cellsexpressing mutant G93A SOD1. Cell survival was determined using theviability stain calcein-AM. At 24 hours, treatment with 100 nM MG132shows no toxicity against any of the cell lines although large numbersof aggregates are seen in the G85R SOD1 cell lines (panel A). Treatmentwith 100 nM MG132 is selectively toxic to the G93A SOD1 cell line after48 hours (panel B). Washing of the cells at 24 hours to remove thecompound does not reverse toxicity to the G93A SOD1 cell line suggestingthat an irreversible toxic event, potentially related the aggregation ofG93A SOD1, has been triggered prior to wash out (panel C).

FIG. 2. Radicicol protects PC12 cells expressing mutant G93A SOD1 fromthe toxic effects of proteasome inhibitor MG132.

FIG. 3. Mutant but not wild type SOD1 aggregates in cells treated withthe proteasome inhibitor MG132. Fluorescence micrographs of PC12 cellsexpressing YFP tagged wild type (WT), G93A mutant (G93A), and G85Rmutant (G85R)SOD1 proteins. The micrographs show the effects of treatingcells with 200 nM MG132 for 24 hours (untreated cells are shown ininsets on left). The wild type SOD1 expressing cells are unaffectedwhile cells expressing mutant SOD1 show large perinuclear aggregates.

FIG. 4. Radicicol decreases mutant SOD1 aggregation induced byproteasome inhibitor MG132. Fluorescence micrographs of PC12 cellsexpressing YFP tagged G93A SOD1 (left) or G85R SOD1 (right) proteins.The cells were untreated, treated with 200 nM MG132 to induce proteinaggregation, or co-treated with MG132 and radicicol for 24 hours.Without radicicol treatment, cells show large perinuclear aggregates.The aggregates are reduced in radicicol-treated cells. While thebehavior of the two cell lines is generally similar, G85R SOD1 cellsshow ‘brighter’ aggregates and more contrast between the aggregates andthe cytoplasm.

FIG. 5. Automated detection of mutant SOD1 aggregates. Left: compoundmicroscope fluorescence micrographs of the same G85R SOD1 cells usingGFP filter set to image the SOD1-YFP aggregates (top) and the TRITCfilter set to image iT-WGA plasma membrane (bottom). Right: Aggregatedetection from the Cellomics Arrayscan 3.5 using spot detector softwareto image cells with Image-iT in channel 1 and SOD1-YFP aggregates inchannel 2. Data are expressed as spot count (aggregates) per object(cell).

FIG. 6. Arylsulfanyl pyrazolones fail to induce heat shock response inHsp 70 promotor assays. HeLa hse-luc cells were treated witharylsulfanyl pyrazolones (1 μM-100 μM), celestrol (1 μM-5 μM), or CdCl2(10 μM-100 μM), for 8 h and activation of the heat shock responsedetermined by HSP70 promoter activity.

FIG. 7. Arylsulfanyl pyrazolones prevent MG132-induced accumulation ofUbi-YFP. HeLa cells were co-transfected with Ubi-YFP and CMV-CFPplasmids, treated with 1 μM MG132 and 25 μM arylsulfanyl pyrazolones andUbi-YFP fluorescence intensity normalized to CFP.

FIG. 8. Brain standard curve for CMB-087229.

FIG. 9. Plasma standard curve for CMB-087229.

FIG. 10. Plasma standard curve for CMB-087229.

FIG. 11. Structures of compounds used in the rat liver microsomalstability study of TC-I-16.

FIG. 12. Minaprine 0-20 minutes HADPH dependent rat liver microsomalstability.

FIG. 13. TC-I-165 0-20 minutes HADPH dependent rat liver microsomalstability.

FIG. 14. Structures of TC-I-165 metabolite candidates (sulfoxidemetabolite candidate TC-II-68 and sulfone metabolite candidateTC-II-70).

FIG. 15. HPLC trace of TC-I-165 [0 min NADPH (tube 1)].

FIG. 16. HPLC trace of TC-I-165 [5 min NADPH (tube 2)] and a new peakwith a retention time of 5.900 minutes.

FIG. 17. HPLC trace of TC-I-165 [60 min NADPH (tube 1)] and a new peakwith a retention time of 5.917 minutes.

FIG. 18. HPLC trace of TC-II-68 [10 μM standard solution (tube 1)] and anew peak with a retention time of 5.933 minutes.

FIG. 19. HPLC trace of TC-II-70 [10 μM standard solution (tube 2)] and anew peak with a retention time of 11.000 minutes.

FIG. 20. Structures of compounds used in the rat liver microsomalstability study of TC-II-70.

FIG. 21. NADPH dependent rat liver microsomal stability study ofTC-II-70.

FIG. 22. NADPH dependent rat liver microsomal stability study ofminaprine.

FIG. 23. NADPH dependent rat liver microsomal stability study ofwarfarin.

FIG. 24. Active arylsulfanyl pyrazolones

FIG. 25. Preliminary SAR of selected arylsulfanylpyrazolones.

FIG. 26. Synthesis of arylsulfanylpyrazolones (Scheme 1).

FIG. 27. Synthesis of arylsulfanylpyrazolones (Scheme 2).

FIG. 28. Kaplan-Meier survival curve for dose response using CMB-087229in G93A SOD1 ALS mice. A 13.4% extension in survival at the highest dose(20 mg/kg) with significance at p<0.05 was observed.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Imbalances in protein homeostasis are often associated with proteinmisfolding and/or protein conformational changes that lead to proteinaggregation and formation of protein inclusion bodies. Manyneurodegenerative diseases, including the polyglutamine (polyQ)-repeatdiseases, Alzheimer's disease, Parkinson's disease, prion diseases, andALS, are characterized by the appearance of damaged and aggregatedproteins, including huntingtin, polyQ proteins, amyloid A prion (PrP andSup35) fibrils, and mutant SOD1 (Taylor et al., Science 2002, 296(5575),1991-1995; Ross, C. A., Neuron. 1997, 19(6), 1147-1150; Perutz, M. F.,Brain Res. Bull. 1999, 50(5-6), 467; and Kopito et al., Nat. Cell Bio.2000, 2(11), E207-E209). The fact that such diverse proteins formaggregates in patients with distinct neurological diseases suggests thata common molecular etiology may contribute to the neuropathology inthese diseases and that, perhaps, protein misfolding and the subsequentappearance of protein aggregates are early events that play a role inneuronal toxicity in multiple human neurological diseases (On, H. T.,Genes. Dev. 2001, 15(8), 925-932; Ikeda et al., Nat. Genet. 1996, 13(2),196-202; DiFiglia et al., Science 1997, 277(5334), 1990-1993; Davies etal., Cell 1997, 90(3), 537-548; and Koo et al., Proc. Natl. Acad. Sci.USA. 1999, 96(18), 9989-9990).

One model for the molecular basis of these neurodegenerative diseases isthat insoluble protein aggregates associate and interfere with theactivity of other critical soluble cellular proteins, and that loss offunction of these diverse proteins has serious negative consequences oncellular function. Affected proteins could include ubiquitin, componentsof the proteasome, components of the cytoskeleton, transcription factors(TBP (i.e., TATA binding protein), EYA (i.e., Eyes Absent protein), CBP(i.e., CREB binding protein), and molecular chaperones Hsc-70, Hsp-70,Hdj-1, and Hdj-2 (Davies et al., Cell 1997, 90(3), 537-548; Ross, C. A.,Neuron. 2002, 35(5), 819-822; Cummings et al., Nat. Genet. 1998, 19(2)148-154; Perez et al., J. Cell Biol. 1998, 143(6), 1457-1470; Kazantsevet al., Proc. Natl. Acad. Sci. USA. 1999, 96(20), 11404-11409; Jana etal., Hum. Mol. Genet. 2001, 10(10), 1049-1059; Nucifora et al., Science2001, 291(5512) 2423-2428; and Suhr et al., J. Cell Biol. 2001, 153(2),283-294). Recent studies showed that TBP and CBP are irreversiblysequestered in polyQ/huntington aggregates, while the chaperone Hsp70 istransiently associated with the surface (Chai et al., Proc. Natl. Acad.Sci. USA. 2002, 99(14), 9310-9315; Kim et al., Nat. Cell. Biol. 2002,4(10), 826-31). Sequestration of CBP into polyglutamine aggregates islinked directly with loss of cellular function in neuronal cells, andoverexpression of CBP suppressed polyQ toxicity (Nucifora et al.,Science 2001, 291(5512) 2423-2428). Furthermore, expression ofpolyglutamine proteins in C. elegans causes other metastable proteins tolose function. Thus, a single aggregation-prone protein may be able todestabilize protein homeostasis in otherwise normal cells (Gidalevitz etal., Science 2006, 311(5766) 1471-1474). These studies indicate that thesequestration of essential soluble cellular proteins in insolubleprotein aggregates could play a significant role in the neuropathologyand neurotoxicity in ALS and related diseases.

It is also possible that the cellular mechanism(s) that remove misfoldedor damaged proteins (Morimoto, R. I., Cell 2002, 110(3), 281-284;Horwich et al., Cell 1997, 89(4), 499-510; and Nollen et al., J. Cell.Sci. 2002, 115(Pt 14) 2809-2816) are overwhelmed in neurodegenerativediseases due to the presence of abundant protein aggregates. Theactivity of molecular chaperones is one of the most important mechanismsto prevent and/or rescue protein misfolding and aggregation. Molecularchaperones are a large and diverse protein family which includes Hsp104,Hsp90, Hsp70, dnaJ (Hsp40), immunophilins (Cyp40, FKBP), Hsp60(chaperonins), the small heat shock proteins, and components of thesteroid aporeceptor complex (p23, Hip, Hop, Bag1) (Gething, M. J.,Nature 1997, 388(6640) 329-331; Bakau, B., Amsterdam: Harwood AcademicPublishers. 1999, 690). They ensure proper protein folding by preventinghydrophobic surfaces from interacting with each other, by enhancingprotein refolding, and, when necessary, by stimulating proteindegradation to remove misfolded proteins that tend to aggregate (Horwichet al., Cell 1997, 89(4), 499-510; Bakau, B., Amsterdam: HarwoodAcademic Publishers. 1999, 690; Schroder et al., Embo. J. 1993, 12(11),4137-4144; Parsell et al., Nature 1994, 372(6505), 475-478; Hartl, F.U., Nature 1996, 381(6583) 571-579; and Morimoto et al., Nat.Biotechnol. 1998, 16(9), 833-838). Accordingly, overexpression ofmolecular chaperones can suppress the toxicity of mutant huntingtin,α-synuclein, and SOD1 (Sakahira et al., Proc. Natl. Acad. Sci. USA.2002, 99 Suppl 4, 6412-6418; Stenoien et al., Hum. Mol. Genet. 1999,8(5), 731-741; Warrick et al., Nat. Genet. 1999, 23(4), 425-428;Carmichael et al., Proc. Natl. Acad. Sci. USA. 2000, 97(17), 9701-9705;Takeuchi et al., Brain Res. 2002, 949(1-2), 11-22; Auluck et al.,Science 2002, 295 (5556), 865-868; and Bailey et al., Hum. Mol. Genet.2002, 11(5), 515-523). Recently, non-chaperone proteins were identifiedthat also suppress toxicity associated with protein aggregation(Kazemi-Esfarjani et al., Science 2000, 287(5459), 1837-1840; andKazemi-Esfarjani et al., Hum. Mol. Genet. 2002, 11(21), 2657-2672).

The chaperone system is a highly appealing therapeutic target, becausemultiple small molecular weight modulators of chaperone activity havealready been identified, two of which are active in a mouse model of ALS(Westerheide et al., J. Biol. Chem. 2005, 280(39), 33097-33100; Kieranet al., Nat. Med. 2004, 10(4), 402-405; and Traynor et al., Neurology2006, 67(1), 20-27). Accordingly, recent analyses identified proteinfolding/misfolding and protein aggregation as a relevant therapeutictarget for neurodegenerative diseases (Pasinelli et al., Nat. Rev.Neurosci. 2006, 7(9), 710-723; Lansbury et al., Nature 2006, 443(7113),774-9; Rubinsztein et al., Nature 2006, 443(7113), 780-786).

Provided Compounds

The present invention provides compounds and methods for treatingpatients with amyotrophic lateral sclerosis (ALS) or otherneurodegenerative diseases characterized by the presence of aberrantprotein aggregates. Without wishing to be bound by any particular theoryor mechanism of action, compounds and methods of the invention areuseful in inhibiting or reversing abnormal protein aggregation orreducing the toxicity of protein aggregation (e.g., SOD1). In certainembodiments, provided compounds are useful in modulating proteosomefunction. The invention provides methods for treating a subject with ALSor other neurodegenerative disease including the step of administeringto the subject a therapeutically effective amount of an inventivecompound or a pharmaceutical composition thereof. In certainembodiments, the subject is a mammal.

In one aspect, the present invention provides compounds of the formula:

or a pharmaceutically acceptable salt thereof, wherein:p is 1-2;

designates a single or double bond, wherein one

is a single bond and one

is a double bond;

-   each R¹ is independently —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R,    —N(R)₂, —CN, —NO₂—NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR,    —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R,    —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or an optionally substituted 3-8    membered saturated, partially unsaturated, or aryl monocyclic ring    having 0-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or an optionally substituted 8-10 membered saturated,    partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; or    -   two R¹ groups are taken together to form

-   -    wherein:        -   X is N or C;        -   each R⁵ is independently —R, —OR, —SR, —S(O)R, —SO₂R,            —OSO₂R, —N(R)₂, —CN, —NO₂—NRC(O)R, —NRC(O)(CO)R,            —NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR,            —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂,            or a 3-8 membered saturated, partially unsaturated, or aryl            monocyclic ring having 0-4 heteroatoms independently            selected from nitrogen, oxygen, or sulfur, or an 8-10            membered saturated, partially unsaturated, or aryl bicyclic            ring having 0-4 heteroatoms independently selected from            nitrogen, oxygen, or sulfur, wherein R⁵ is optionally            substituted with 1-5 R groups;

-   each R is independently hydrogen, halogen, optionally substituted    C₁₋₂₀ aliphatic, optionally substituted C₁₋₂₀ heteroaliphatic,    optionally substituted phenyl, or optionally substituted arylalkyl,    or two R on the same nitrogen are taken together to form a 5-6    membered saturated, partially saturated, or aryl ring having 1-3    heteroatoms independently selected from nitrogen, oxygen, and    sulfur, or two R on the same carbon or on adjacent carbons are    optionally taken together to form a 3-6 membered saturated    cycloalkyl or fused monocyclic ring containing 0-2 heteroatoms    independently selected from nitrogen, oxygen, and sulfur;

-   n is 0-1;

-   each R² and R³ are independently —R, —OR, —SR, —S(O)R, —SO₂R,    —OSO₂R, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂,    a suitable amino protecting group, or an optionally substituted 3-8    membered saturated, partially unsaturated, or aryl monocyclic ring    having 0-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or an optionally substituted 8-10 membered saturated,    partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;

-   R⁰ is R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, N(R)₂, —NRC(O)R,    —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R,    —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂,    —OC(O)N(R)₂, or

-    wherein:    -   L is a valence bond or a bivalent saturated or partially        unsaturated C₁₋₁₀ hydrocarbon chain, wherein 1-4 methylene units        of L are optionally and independently replaced by —O—, —S—,        —C(O)—, —OC(O)—, —C(O)O—, —OC(O)O—, —S(O)—, —S(O)₂—, —OSO₂O—,        —N(R)C(O)—, —C(O)NR—, —N(R)C(O)O—, —OC(O)NR—, —N(R)C(O)NR—,        —N(R⁶)—, or -Cy-, and wherein L is optionally substituted with        1-4 R groups; wherein:        -   each -Cy- is independently a bivalent optionally substituted            saturated, partially unsaturated, or aromatic monocyclic or            bicyclic ring selected from a 6-10 membered arylene, a 5-10            membered heteroarylene having 1-4 heteroatoms independently            selected from oxygen, nitrogen, or sulfur, a 3-8 membered            carbocyclylene, or a 3-10 membered heterocyclylene having            1-4 heteroatoms independently selected from oxygen,            nitrogen, or sulfur;        -   R⁶ is —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —C(O)R, —C(O)OR,            —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, a suitable amino            protecting group, or an optionally substituted 3-8 membered            saturated, partially unsaturated, or aryl monocyclic ring            having 0-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, or an optionally substituted 8-10            membered saturated, partially unsaturated, or aryl bicyclic            ring having 0-4 heteroatoms independently selected from            nitrogen, oxygen, or sulfur;    -   Ring A is a 3-8 membered saturated, partially unsaturated, or        aryl monocyclic ring optionally containing 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur, or an        8-10 membered saturated, partially unsaturated, or aryl bicyclic        ring optionally containing 0-4 heteroatoms independently        selected from nitrogen, oxygen, or sulfur, and wherein Ring A is        optionally substituted with m occurrences of R⁴;        -   m is 0-5; and    -   each R⁴ is independently —R, —OR, —SR, —CN, —S(O)R, —SO₂R,        —OSO₂R, —N(R)₂, —NO₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂,        —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR,        —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or a 3-8 membered        saturated, partially unsaturated, or aryl monocyclic ring        optionally containing 0-4 heteroatoms independently selected        from nitrogen, oxygen, or sulfur.

In certain embodiments, n is 0. In certain embodiments, n is 1.

In certain embodiments, p is 1. In certain embodiments, p is 2.

As described above and herein,

designates a single or double bond. It will be understood by one ofordinary skill in the art that when one

designates a double bond between two carbons to provide a compound offormula Ia:

then n is 1 and p is 1. Similarly, when

designates a double bond between a carbon and a nitrogen to provide acompound of formula Ib:

then n is 0 and p is 1 or 2, depending on the tautomeric form of 1b,e.g., in compounds of formula 1b wherein at least one R¹ is hydrogen,provided compounds may exist in any tautomeric form available. One suchexemplary pair of tautomers is as shown below:

As defined generally above, each R¹ is independently —R, —OR, —SR,—S(O)R, —SO₂R, —OSO₂R, —N(R)₂, —CN, —NO₂, —NRC(O)R, —NRC(O)(CO)R,—NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R,—C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or an optionallysubstituted 3-8 membered saturated, partially unsaturated, or arylmonocyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an optionally substituted 8-10 memberedsaturated, partially unsaturated, or aryl bicyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur; or

two R¹ groups are taken together to form

In some embodiments, each R¹ is independently —R, —OR, —SR, —S(O)R,—SO₂R, —OSO₂R, N(R)₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR,—N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR,—C(O)N(R)₂, or —OC(O)N(R)₂.

In some embodiments, at least one R¹ is optionally substituted 3-8membered saturated monocyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, at leastone R¹ is an optionally substituted 3-6 membered saturated monocyclicring having 0-2 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, at least one R¹ is an optionallysubstituted 5-6 membered saturated monocyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, at least one R¹ is an optionally substituted 3-8membered partially unsaturated monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, at least one R¹ is an optionally substituted 3-6 memberedpartially unsaturated monocyclic ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, at least one R¹ is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, at least one R¹ is an optionally substituted 5-6membered aryl ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, at least one R¹ is anoptionally substituted 5-6 membered aryl ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, at least one R¹ is an optionally substituted 5 memberedaryl ring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, at least one R¹ is an optionallysubstituted 6 membered aryl ring having 1-3 nitrogens. In someembodiments, at least one R¹ is an optionally substituted phenyl. Incertain embodiments, at least one R¹ is unsubstituted phenyl.

In some embodiments, at least one R¹ is an optionally substituted 8-10membered saturated bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, at leastone R¹ is an optionally substituted 8-10 membered saturated bicyclicring having 0-2 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, at least one R¹ is an optionallysubstituted 8-10 membered saturated bicyclic carbocycle.

In some embodiments, at least one R¹ is an optionally substituted 8-10membered partially unsaturated bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, at least one R¹ is an optionally substituted 8-10 memberedpartially unsaturated bicyclic ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, at leastone R¹ is an optionally substituted 8-10 membered partially unsaturatedcarbocycle.

In some embodiments, at least one R¹ is an optionally substituted 9-10membered aryl ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, at least one R¹ is anoptionally substituted 9-10 membered aryl ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, at least one R¹ is an optionally substituted 9 memberedaryl ring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, at least one R¹ is an optionallysubstituted 10 membered aryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, at leastone R¹ is optionally substituted naphthyl.

In some embodiments, two R¹ are taken together to form

wherein X is N or C. In certain embodiments wherein X is N, one R⁵ groupis present. In certain embodiments wherein X is C, either one or two R⁵groups may be present. In certain embodiments, two R¹ are taken togetherto form any one of the following formulae:

In some embodiments, each R⁵ is independently —R, —OR, —SR, —S(O)R,—SO₂R, —OSO₂R, —N(R)₂, —CN, —NO₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂,—NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R,—OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂. In some embodiments, at least one R⁵is a 3-8 membered saturated, partially unsaturated, or aryl monocyclicring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated,or aryl bicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein R⁵ is optionally substituted with1-5 R groups.

As defined generally above and herein, R² is independently —R, —OR, —SR,—S(O)R, —SO₂R, —OSO₂R, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂,—OC(O)N(R)₂, a suitable amino protecting group, or an optionallysubstituted 3-8 membered saturated, partially unsaturated, or arylmonocyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an optionally substituted 8-10 memberedsaturated, partially unsaturated, or aryl bicyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R² is —R, —OR, —SR, —N(R)₂, or a suitable aminoprotecting group. In certain embodiments, R² is halogen or R. In certainembodiments, R² is hydrogen, methyl, ethyl, propyl, or butyl. In certainembodiments, R² is hydrogen. In certain embodiments, R² is methyl.

In some embodiments, R² is an optionally substituted 3-8 memberedsaturated monocyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R² is anoptionally substituted 3-6 membered saturated monocyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R² is an optionally substituted 5-6 membered saturatedmonocyclic ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R² is an optionally substituted 3-8 memberedpartially unsaturated monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R² is an optionally substituted 3-6 membered partiallyunsaturated monocyclic ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R² is anoptionally substituted 5-6 membered partially unsaturated monocyclicring having 0-2 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, R² is an optionally substituted 5-6 membered arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R² is an optionally substituted5-6 membered aryl ring having 0-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R² is anoptionally substituted 5 membered aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R² is an optionally substituted 6 membered aryl ring having1-3 nitrogens. In some embodiments, R² is an optionally substitutedphenyl. In certain embodiments, R² is unsubstituted phenyl.

In some embodiments, R² is an optionally substituted 8-10 memberedsaturated bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R² is anoptionally substituted 8-10 membered saturated bicyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R² is an optionally substituted 8-10 memberedsaturated bicyclic carbocycle.

In some embodiments, R² is an optionally substituted 8-10 memberedpartially unsaturated bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R² is anoptionally substituted 8-10 membered partially unsaturated bicyclic ringhaving 0-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R² is an optionally substituted 8-10membered partially unsaturated carbocycle.

In some embodiments, R² is an optionally substituted 9-10 membered arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R² is an optionally substituted9-10 membered aryl ring having 0-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R² is anoptionally substituted 9 membered aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R² is an optionally substituted 10 membered aryl ringhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R² is optionally substituted naphthyl.

Exemplary optionally substituted R² heteroaryl groups include thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, orchromanyl.

As defined generally above and herein, R³ is independently —R, —OR, —SR,—S(O)R, —SO₂R, —OSO₂R, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂,—OC(O)N(R)₂, a suitable amino protecting group, or an optionallysubstituted 3-8 membered saturated, partially unsaturated, or arylmonocyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an optionally substituted 8-10 memberedsaturated, partially unsaturated, or aryl bicyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R³ is —R, —OR, —SR, —N(R)₂, or a suitable aminoprotecting group. In certain embodiments, R³ is halogen or R. In certainembodiments, R³ is hydrogen, methyl, ethyl, propyl, or butyl. In certainembodiments, R³ is hydrogen. In certain embodiments, R³ is methyl.

In some embodiments, R³ is an optionally substituted 3-8 memberedsaturated monocyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R³ is anoptionally substituted 3-6 membered saturated monocyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R³ is an optionally substituted 5-6 membered saturatedmonocyclic ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R³ is an optionally substituted 3-8 memberedpartially unsaturated monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R³ is an optionally substituted 3-6 membered partiallyunsaturated monocyclic ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R³ is anoptionally substituted 5-6 membered partially unsaturated monocyclicring having 0-2 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, R³ is an optionally substituted 5-6 membered arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R³ is an optionally substituted5-6 membered aryl ring having 0-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R³ is anoptionally substituted 5 membered aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R³ is an optionally substituted 6 membered aryl ring having1-3 nitrogens. In some embodiments, R³ is an optionally substitutedphenyl. In certain embodiments, R³ is unsubstituted phenyl.

In some embodiments, R³ is an optionally substituted 8-10 memberedsaturated bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R³ is anoptionally substituted 8-10 membered saturated bicyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R³ is an optionally substituted 8-10 memberedsaturated bicyclic carbocycle.

In some embodiments, R³ is an optionally substituted 8-10 memberedpartially unsaturated bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R³ is anoptionally substituted 8-10 membered partially unsaturated bicyclic ringhaving 0-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R³ is an optionally substituted 8-10membered partially unsaturated carbocycle.

In some embodiments, R³ is an optionally substituted 9-10 membered arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R³ is an optionally substituted9-10 membered aryl ring having 0-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R³ is anoptionally substituted 9 membered aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R³ is an optionally substituted 10 membered aryl ringhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R³ is optionally substituted naphthyl.

Exemplary optionally substituted R³ heteroaryl groups include thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, orchromanyl.

In certain embodiments, R³ is of any one of the following formulae:

As described generally above and herein, R⁰ is —R—OR, —SR, —S(O)R,—SO₂R, —OSO₂R, —N(R)₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR,—N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR,—C(O)N(R)₂, —OC(O)N(R)₂, or

In some embodiments, R⁰ is R, wherein R is hydrogen, optionallysubstituted C₁₋₆ aliphatic, optionally substituted C₁₋₆ heteroaliphatic,or optionally substituted phenyl. In certain embodiments, R⁰ is methyl,ethyl, propyl, or butyl.

In some embodiments, R⁰ is

wherein R⁴, m, and Ring A are as defined above and L is a valence bond.In some embodiments, R⁰ is

wherein R⁴, m, and Ring A are as defined above and L is not a valencebond.

As defined generally above, L is a valence bond or a bivalent saturatedor partially unsaturated C₁₋₁₀ hydrocarbon chain, wherein 1-4 methyleneunits of L are optionally and independently replaced by —O—, —S—,—C(O)—, —OC(O)—, —C(O)O—, —OC(O)O—, —S(O)—, —S(O)₂—, —OSO₂O—,—N(R)C(O)—, —C(O)NR—, —N(R)C(O)O—, —OC(O)NR—, —N(R)C(O)NR—, —N(R⁶)—, or-Cy-, and wherein L is optionally substituted with 1-4 R groups;wherein:

-   -   each -Cy- is independently a bivalent optionally substituted        saturated, partially unsaturated, or aromatic monocyclic or        bicyclic ring selected from a 6-10 membered arylene, a 5-10        membered heteroarylene having 1-4 heteroatoms independently        selected from oxygen, nitrogen, or sulfur, a 3-8 membered        carbocyclylene, or a 3-10 membered heterocyclylene having 1-4        heteroatoms independently selected from oxygen, nitrogen, or        sulfur;    -   R⁶ is —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —C(O)R, —C(O)OR,        —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, a suitable amino        protecting group, or an optionally substituted 3-8 membered        saturated, partially unsaturated, or aryl monocyclic ring having        0-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or an optionally substituted 8-10 membered saturated,        partially unsaturated, or aryl bicyclic ring having 0-4        heteroatoms independently selected from nitrogen, oxygen, or        sulfur.

In some embodiments, L a bivalent saturated or partially unsaturatedC₁₋₁₀ hydrocarbon chain, wherein 1-4 methylene units of L are optionallyand independently replaced by —O—, —N(R⁶)—, —S—, —C(O)—, —OC(O)—,—C(O)O—, —OC(O)O—, —S(O)—, —S(O)₂—, —OSO₂O—, —N(R)C(O)—, —C(O)NR—,—N(R)C(O)O—, —OC(O)NR—, —N(R)C(O)NR—, and wherein L is optionallysubstituted with 1-4 R groups. In some embodiments, L a bivalentsaturated or partially unsaturated C₂₋₇ hydrocarbon chain, wherein 1-3methylene units of L are optionally and independently replaced by—N(R⁶)—, —N(R)C(O)—, —C(O)N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—,—S(O)— or —SO₂—, and wherein L is optionally substituted with 1-4 Rgroups. In some embodiments, L is a bivalent saturated C₂₋₇ hydrocarbonchain, wherein one or more methylene units of L is replaced by —N(R⁶)—,—N(R)C(O)—, —C(O)N(R)—. In some embodiments, L is a bivalent saturatedC₂₋₇ hydrocarbon chain, wherein one or more methylene units of L isreplaced by —O—, —C(O)—, —OC(O)—, or —C(O)O—. In some embodiments, L isa bivalent saturated C₂₋₇ hydrocarbon chain, wherein one or moremethylene units of L is replaced by —S—, —S(O)— or —SO₂—.

In certain embodiments, L is of any of the following formulae:

In certain embodiments, L is of any of the following formulae:

In certain embodiments, L is of any of the following formulae:

In some embodiments, L is a bivalent saturated C₁₋₁₀ hydrocarbon chain,wherein one or more methylene unit is independently replaced by -Cy-,and wherein one or more -Cy- is independently a bivalent optionallysubstituted saturated monocyclic ring. In some embodiments, one or more-Cy- is independently a bivalent optionally substituted partiallyunsaturated monocyclic ring. In some embodiments, one or more -Cy- isindependently a bivalent optionally substituted aromatic monocyclicring. In certain embodiments, -Cy- is optionally substituted phenylene.

In some embodiments, one or more -Cy- is independently a bivalentoptionally substituted saturated bicyclic ring. In some embodiments, oneor more -Cy- is independently a bivalent optionally substitutedpartially unsaturated bicyclic ring. In some embodiments, one or more-Cy- is independently a bivalent optionally substituted aromaticbicyclic ring. In certain embodiments, -Cy- is optionally substitutednaphthylene.

In some embodiments, one or more -Cy- is independently an optionallysubstituted 6-10 membered arylene. In some embodiments, one or more -Cy-is independently an optionally substituted a 5-10 membered heteroarylenehaving 1-4 heteroatoms independently selected from oxygen, nitrogen, orsulfur. In some embodiments, one or more -Cy- is independently anoptionally substituted a 5-6 membered heteroarylene having 1-4heteroatoms independently selected from oxygen, nitrogen, or sulfur. Insome embodiments, one or more -Cy- is independently an optionallysubstituted 5 membered heteroarylene having 1-4 heteroatomsindependently selected from oxygen, nitrogen, or sulfur. In someembodiments, one or more -Cy- is independently an optionally substituted6 membered heteroarylene having 1-4 heteroatoms independently selectedfrom oxygen, nitrogen, or sulfur.

Exemplary optionally substituted -Cy- heteroarylene groups includethienylene, furanylene, pyrrolylene, imidazolylene, pyrazolylene,triazolylene, tetrazolylene, oxazolylene, isoxazolylene, oxadiazolylene,thiazolylene, isothiazolylene, thiadiazolylene, pyridylene,pyridazinylene, pyrimidinylene, pyrazinylene, indolizinylene,purinylene, naphthyridinylene, pteridinylene, indolylene, isoindolylene,benzothienylene, benzofuranylene, dibenzofuranylene, indazolylene,benzimidazolylene, benzthiazolylene, quinolylene, isoquinolylene,cinnolinylene, phthalazinylene, quinazolinylene, quinoxalinylene,4H-quinolizinylene, carbazolylene, acridinylene, phenazinylene,phenothiazinylene, phenoxazinylene, tetrahydroquinolinylene,tetrahydroisoquinolinylene, pyrido[2,3-b]-1,4-oxazin-3(4H)-onylene, andchromanylene.

In certain embodiments, -Cy- is selected from the group consisting oftetrahydropyranylene, tetrahydrofuranylene, morpholinylene,thiomorpholinylene, piperidinylene, piperazinylene, pyrrolidinylene,tetrahydrothiophenylene, and tetrahydrothiopyranylene, wherein each ringis optionally substituted.

In some embodiments, one or more -Cy- is independently an optionallysubstituted 3-8 membered carbocyclylene. In some embodiments, one ormore -Cy- is independently an optionally substituted 3-6 memberedcarbocyclylene. In some embodiments, one or more -Cy- is independentlyan optionally substituted cyclopropylene, cyclopentylene, orcyclohexylene.

In some embodiments, one or more -Cy- is independently an optionallysubstituted 3-10 membered heterocyclylene having 1-4 heteroatomsindependently selected from oxygen, nitrogen, or sulfur. In someembodiments, one or more -Cy- is independently an optionally substituted5-7 membered heterocyclylene having 1-3 heteroatoms independentlyselected from oxygen, nitrogen, or sulfur. In some embodiments, one ormore -Cy- is independently an optionally substituted 3 memberedheterocyclylene having 1 heteroatom independently selected from oxygen,nitrogen, or sulfur. In some embodiments, one or more -Cy- isindependently an optionally substituted 5 membered heterocyclylenehaving 1-2 heteroatoms independently selected from oxygen, nitrogen, orsulfur. In some embodiments, one or more -Cy- is independently anoptionally substituted 6 membered heterocyclylene having 1-3 heteroatomsindependently selected from oxygen, nitrogen, or sulfur.

Exemplary -Cy- saturated 3-8 membered optionally substitutedheterocyclenes include oxiranylene, oxetanylene, tetrahydrofuranylene,tetrahydropyranylene, oxepaneylene, aziridineylene, azetidineylene,pyrrolidinylene, piperidinylene, azepanylene, thiiranylene,thietanylene, tetrahydrothiophenylene, tetrahydrothiopyranylene,thiepanylene, dioxolanylene, oxathiolanylene, oxazolidinylene,imidazolidinylene, thiazolidinylene, dithiolanylene, dioxanylene,morpholinylene, oxathianylene, piperazinylene, thiomorpholinylene,dithianylene, dioxepanylene, oxazepanylene, oxathiepanylene,dithiepanylene, diazepanylene, dihydrofuranonylene,tetrahydropyranonylene, oxepanonylene, pyrrolidinonylene,piperidinonylene, azepanonylene, dihydrothiophenonylene,tetrahydrothiopyranonylene, thiepanonylene, oxazolidinonylene,oxazinanonylene, oxazepanonylene, dioxolanonylene, dioxanonylene,dioxepanonylene, oxathiolinonylene, oxathianonylene, oxathiepanonylene,thiazolidinonylene, thiazinanonylene, thiazepanonylene,imidazolidinonylene, tetrahydropyrimidinonylene, diazepanonylene,imidazolidinedionylene, oxazolidinedionylene, thiazolidinedionylene,dioxolanedionylene, oxathiolanedionylene, piperazinedionylene,morpholinedionylene, and thiomorpholinedionylene.

In some embodiments, R⁶ is —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —C(O)R,—C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, or —OC(O)N(R)₂. In someembodiments, R⁶ is a suitable amino protecting group. In certainembodiments, R⁶ is —SO₂R. In some embodiments, R⁶ is R. In someembodiments, R⁶ is optionally substituted arylalkyl.

In some embodiments, R⁶ is an optionally substituted 3-8 memberedsaturated monocyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁶ is anoptionally substituted 3-6 membered saturated monocyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁶ is an optionally substituted 5-6 membered saturatedmonocyclic ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R⁶ is an optionally substituted 3-8 memberedpartially unsaturated monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁶ is an optionally substituted 3-6 membered partiallyunsaturated monocyclic ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁶ is anoptionally substituted 5-6 membered partially unsaturated monocyclicring having 0-2 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, R⁶ is an optionally substituted 5-6 membered arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R⁶ is an optionally substituted5-6 membered aryl ring having 0-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁶ is anoptionally substituted 5 membered aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁶ is an optionally substituted 6 membered aryl ring having1-3 nitrogens. In some embodiments, R⁶ is an optionally substitutedphenyl. In certain embodiments, R⁶ is unsubstituted phenyl.

In some embodiments, R⁶ is an optionally substituted 8-10 memberedsaturated bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁶ is anoptionally substituted 8-10 membered saturated bicyclic ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁶ is an optionally substituted 8-10 memberedsaturated bicyclic carbocycle.

In some embodiments, R⁶ is an optionally substituted 8-10 memberedpartially unsaturated bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁶ is anoptionally substituted 8-10 membered partially unsaturated bicyclic ringhaving 0-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R⁶ is an optionally substituted 8-10membered partially unsaturated carbocycle.

In some embodiments, R⁶ is an optionally substituted 9-10 membered arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R⁶ is an optionally substituted9-10 membered aryl ring having 0-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁶ is anoptionally substituted 9 membered aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁶ is an optionally substituted 10 membered aryl ringhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R⁶ is optionally substituted naphthyl.

Exemplary optionally substituted R⁶ heteroaryl groups include thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, orchromanyl.

In certain embodiments, L is a bivalent saturated C₂₋₇ hydrocarbon chainwherein two methylene units are replaced by —N(R⁶)—, —N(R)C(O)—,—C(O)N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)— or —SO₂—. Incertain embodiments, L is a bivalent saturated C₂₋₇ hydrocarbon chainwherein two methylene units are replaced by —O—, —S—, —S(O)—, or —SO₂—.In certain embodiments, L is a bivalent saturated C₂ hydrocarbon chainwherein at least one methylene unit is replaced by —N(R⁶)—, —N(R)C(O)—,—C(O)N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —S(O)— or —SO₂—. Incertain embodiments, L is a bivalent saturated C₂ hydrocarbon chainwherein at least one methylene unit is replaced by —N(R⁶)—, —O—, —S—,—S(O)—, or —SO₂—.

In certain embodiments, L is of any of the following formulae:

As described generally above and herein, Ring A is a 3-8 memberedsaturated, partially unsaturated, or aryl monocyclic ring optionallycontaining 0-4 heteroatoms independently selected from nitrogen, oxygen,or sulfur, or an 8-10 membered saturated, partially unsaturated, or arylbicyclic ring optionally containing 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, and wherein Ring A isoptionally substituted with m occurrences of R⁴, wherein m is 0-5. Insome embodiments, m is 0, 1, 2, or 3.

In certain embodiments, Ring A is phenyl substituted with 1-5 R⁴ groups.In certain embodiments, Ring A is unsubstituted phenyl.

In certain embodiments, Ring A is naphthyl substituted with 1-5 R⁴groups. In certain embodiments, Ring A is unsubstituted naphthyl.

In some embodiments, Ring A is a 5-6 membered monocyclic saturated,partially unsaturated or aromatic heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, andoptionally substituted with 1-5 R⁴ groups. In some embodiments, Ring Ais a 5 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and optionallysubstituted with 1-2 R⁴ groups. In other embodiments, Ring A is a 6membered monocyclic heteroaryl ring having 1-2 nitrogens independentlyselected from nitrogen, oxygen, or sulfur, and optionally substitutedwith 1-2 R⁴ groups.

In certain embodiments, Ring A is an 8-10 membered bicyclic ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,and optionally substituted with 1-5 R⁴ groups. In some embodiments, RingA is an 8 membered bicyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, and optionally substitutedwith 1-3 R⁴ groups. In some embodiments, Ring A is a 9 membered bicyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur, and optionally substituted with 1-3 R⁴ groups. Insome embodiments, Ring A is a 10 membered bicyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andoptionally substituted with 1-3 R⁴ groups. In some embodiments, Ring Ais an 8-10 membered bicyclic ring comprised of 0-2 aromatic rings andoptionally substituted with 1-5 R⁴ groups.

Exemplary Ring A heteroaryl groups include thienyl, furanyl, pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, orchromanyl, wherein Ring A is optionally substituted with 1-5 R⁴ groups.

As described generally above and herein, each R⁴ is independently —R,—OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —N(R)₂, —CN, —NO₂, —NRC(O)R,—NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R,—N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂,or a 3-8 membered saturated, partially unsaturated, or aryl monocyclicring optionally containing 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, at least one R⁴ is independently —OR, —SR, —S(O)R,—SO₂R, —OSO₂R, N(R)₂, —NO₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂,—NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R,—OC(O)OR, —C(O)N(R)₂, or —OC(O)N(R)₂. In certain embodiments, m is 1 andR⁴ is —OR. In certain embodiments, m is 1 and R⁴ is —OMe. In certainembodiments, m is 1 and R⁴ is —NO₂. In certain embodiments, m is 1 andR⁴ is —NR₂. In certain embodiments, m is 1 and R⁴ is —NMe₂.

In some embodiments, at least one R⁴ is independently a 3-8 memberedsaturated, partially unsaturated, or aryl monocyclic ring optionallycontaining 0-4 heteroatoms independently selected from nitrogen, oxygen,or sulfur.

In some embodiments, at least one R⁴ is independently a 3-6 memberedsaturated monocyclic ring optionally containing 0-1 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, at least one R⁴ is independently a 3 membered saturatedmonocyclic ring optionally containing 0-1 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, atleast one R⁴ is cyclopropyl. In certain embodiments, two R⁴ arecyclopropyl.

In certain embodiments, at least one R⁴ is optionally substitutedphenyl. In certain embodiments, at least one R⁴ is unsubstituted phenyl.In some embodiments, at least one R⁴ is independently a 5-6 memberedaryl monocyclic ring containing 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In certain embodiments, R⁴ is of any of the following formulae:

In some embodiments, each R⁴ is R, wherein R is hydrogen, halogen, oroptionally substituted C₁₋₂₀ aliphatic. In certain embodiments, each R⁴is independently C₁₋₆ aliphatic. In certain embodiments, each R⁴ isindependently selected from the group consisting of methyl, ethyl,propyl, or butyl. In certain embodiments, m is 1 and R⁴ is methyl,ethyl, propyl, or butyl. In certain embodiments, m is 1 and R⁴ is C₁₄aliphatic. In some embodiments, m is 2 and each R⁴ is methyl. In someembodiments, m is 2 and each R⁴ is —CF₃.

In certain embodiments, each R⁴ is independently selected from the groupconsisting of fluorine, chlorine, bromine, and iodine. In certainembodiments, m is 1 and R⁴ is fluorine. In certain embodiments, m is 1and R⁴ is chlorine. In certain embodiments, m is 1 and R⁴ is bromine. Incertain embodiments, m is 2 and each R⁴ is fluorine. In certainembodiments, m is 2 and each R⁴ is chlorine. In certain embodiments, mis 2 and each R⁴ is bromine. In certain embodiments, m is 2, wherein oneR⁴ is fluorine and one R⁴ is chlorine. In certain embodiments, m is 3and each R⁴ is independently methyl or chlorine.

In certain embodiments, Ring A is of any one of the formulae:

wherein R⁴ is as defined above and herein.

In certain embodiments, Ring A is of any one of the formulae:

In certain embodiments, Ring A is of either one of the formulae:

In certain embodiments, Ring A is of the formula:

In certain embodiments, Ring A is of any one of the formulae:

wherein R⁴ is as defined above and herein.

In certain embodiments, Ring A is of the formula:

In certain embodiments, Ring A is of any one of the formulae:

wherein R⁴ is as defined above and herein.

In certain embodiments, Ring A is of either of the formulae:

In certain embodiments, Ring A is of any one of the formulae:

wherein R⁴ is as defined above and herein.

In certain embodiments, Ring A of the formula:

wherein R⁴ is as defined above and herein.

In some embodiments, a provided compound is of either of the formulae:

wherein R¹, R², R³, R⁴, L, m, and Ring A are as defined and describedabove and herein.

In some embodiments, a provided compound is of either of the formulae:

wherein R¹, R², R³, R⁴, L and m are as defined and described above andherein. In certain embodiments, wherein compounds are of formula 1a(i-a)or 1b(i-a), m is 1, 2, or 3. In certain embodiments, wherein compoundsare of formula 1a(i-a) or 1b(i-a), m is 1 and R⁴ is meta to L. Incertain embodiments, wherein compounds are of formula 1a(i-a) or1b(i-a), m is 2 and each R⁴ is meta to L.

In certain embodiments, wherein compounds are of formula 1a(i-a) or1b(i-a), L is not a bivalent saturated C₂ hydrocarbon chain wherein onemethylene unit is replaced with —S(O)—. In certain embodiments, whereincompounds are of formula 1a(i-a) or 1b(i-a), L is a bivalent saturatedC₂ hydrocarbon chain wherein one methylene unit is replaced with —S—,—O—, or —SO₂—.

In certain embodiments, wherein compounds are of formula 1a(i-a) or1b(i-a), at least one of R¹, R², or R³ is hydrogen. In certainembodiments, wherein compounds are of formula 1a(i-a) or 1b(i-a), atleast two of R¹, R², and/or R³ are hydrogen. In certain embodiments,wherein compounds are of formula 1a(i-a) or 1b(i-a), R¹, R², and R³ areall hydrogen.

In certain embodiments, a provided compound is of the formula:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In certain embodiments, a provided compound is of the formula:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In certain embodiments, a provided compound is of the formula:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In certain embodiments, a provided compound is of the formula:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of the formula:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of the formula:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of the formula:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein. In certain embodiments, R₄ is halogen or C₁-C₆ alkyl.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of the formula:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of the formula:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

In certain embodiments, a provided compound is the formula:

wherein R¹, R², R³, R⁴, R⁶ and m are as defined and described above andherein.

In certain embodiments, a provided compound is the formula:

wherein R, R¹, R², R³, R⁴, and m are as defined and described above andherein.

In certain embodiments, a provided compound is the formula:

wherein R, R¹, R², R³, R⁴, and m are as defined and described above andherein.

In certain embodiments, a provided compound is the formula:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, R⁴, R⁶ and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R, R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R, R¹, R², R³, R⁴, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, R⁴, and m are as defined and described above andherein.

In certain embodiments, a provided compound is of the formula:

wherein R¹, R², R³, R⁴ and R⁶ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R¹, R², R³, and R⁴ are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of any one of the formulae:

In some embodiments, a provided compound is of either of the formulae:

wherein R³, R⁴, R⁵, m, L, and Ring A are as defined and described aboveand herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein each R⁵ is independently —R. In certainembodiments, a provided compound is of formula 1b(i-b) or 1b(i-c) above,wherein each R⁵ is independently —OR, —SR, —S(O)R, —SO₂R, —OSO₂R,—N(R)₂, —CN, —NO₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR,—N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR,—C(O)N(R)₂, or —OC(O)N(R)₂. In certain embodiments, R⁵ is —NMe₂.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein at least one R₅ is a 3-8 membered saturated,partially unsaturated, or aryl monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein R⁵ isoptionally substituted with 1-5 R groups. In certain embodiments, atleast one R₅ is a 5-6 membered aryl monocyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein R⁵ is optionally substituted with 1-5 R groups. In certainembodiments, at least one R⁵ is phenyl optionally substituted with 1-5 Rgroups.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein at least one R⁵ is a 5-6 membered heteroarylmonocyclic ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, and wherein R⁵ is optionally substitutedwith 1-5 R groups.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ is an 8-10 membered saturated, partiallyunsaturated, or aryl bicyclic ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein R⁵ is optionallysubstituted with 1-5 R groups.

Exemplary R⁵ heteroaryl groups include thienyl, furanyl, pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, orchromanyl, wherein R⁵ is optionally substituted with 1-5 R groups.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ is of the formula:

wherein R is as defined and described above and herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ is of any of the formulae:

wherein R is as defined and described above and herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ is of any of the formulae:

wherein R is as defined and described above and herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ is of any of the formulae:

wherein R is as defined and described above and herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ is of any of the formulae:

wherein R is as defined and described above and herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ of the formula:

wherein R is as defined and described above and herein.

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ of any of the formulae:

In certain embodiments, a provided compound is of the formula:

In certain embodiments, a provided compound is of formula 1b(i-b) or1b(i-c) above, wherein R⁵ of any of the formulae:

wherein R, R³, R⁴, L, and X are as defined and described above andherein.

In certain embodiments, a provided compound is of the formula:

wherein R, R³, R⁴, L, and X are as defined and described above andherein.

In certain embodiments, a provided compound is of the formula:

wherein R, R³, R⁴, L, and X are as defined and described above andherein.

In certain embodiments, a provided compound is of the formula:

wherein R, R³, R⁴, L, X, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein L and X are as defined and described above and herein.

In some embodiments, a provided compound is of any one of the formulae:

In certain embodiments, a provided compound is of the formula:

In certain embodiments, a provided compound is of the formula:

In certain embodiments, a provided compound is of the formula:

In certain embodiments, a provided compound is of the formula:

In certain embodiments, a provided compound is of the formula:

wherein R, R³, R⁴, L, and m are as defined and described above andherein.

In some embodiments, a provided compound is of any one of the formulae:

wherein R³ and R⁴ are as defined and described above and herein, andwherein L is selected from the group consisting of —CH₂O—, —CH₂S—,—CH₂S(O)—, or —CH₂SO₂—.

In some embodiments, a provided compound is of any one of the formulae:

wherein L is selected from the group consisting of —CH₂O—, —CH₂S—,—CH₂S(O)—, or —CH₂SO₂—.

In certain embodiments, a provided compound is of the formula:

In some embodiments, a provided compound is of the formula:

wherein R, R³, R⁴, and m are as defined and described above and herein,and wherein L is selected from the group consisting of —CH₂O—, —CH₂S—,—CH₂S(O)—, or —CH₂SO₂—.

Exemplary compounds of the present invention having an EC₅₀ of <20 μM inassays for protection of mutant SOD1-induced cytotoxicity are as shownbelow in Table 1:

TABLE 1

Methods of Treating Amyotrophic Lateral Sclerosis (ALS)

The present invention provides methods of treating ALS comprisingadministering a therapeutically effective amount of a provided compoundor analog thereof, or pharmaceutical composition thereof, to a subjectwith ALS. The inventive pyrazolone may be administered to a subject inneed thereof using any method of administration known in the medicalarts. In certain embodiments, treatment may be administered orally orparenterally. In some embodiments, treatment is administered once a day.In some embodiments, the compound is administered two, three, four, orfive times a day. In some embodiments, the compound is administeredevery other day. In some embodiments, the compound is administered everytwo days. In some embodiments, the compound is administered every threedays. In some embodiments, the compound is administered every four days.In some embodiments, the compound is administered every five days. Insome embodiments, the compound is administered every six days. In someembodiments, the compound is administered once a week. In someembodiments, the compound is administered at intervals as instructed bya physician for the duration of the life of the patient being treated.In certain embodiments, treatment is administered as many times a day asnecessary to provide a therapeutically effective amount of a providedcompound to treat a subject with ALS. In some embodiments, the subjectwith ALS is a mammal. In some embodiments, the subject with ALS is arodent, such as a rat or mouse, for example, a mouse model of ALS. Incertain embodiments, the subject with ALS is a human.

The efficacy of the inventive treatment may be evaluated and followedusing any method known in the medical arts. The treatment of ALS may beevaluated, for example, by physical examination, laboratory testing,imaging studies, electrophysiological studies, etc.

Method of Inhibiting or Reversing Abnormal Protein Aggregation

The present invention provides a method of inhibiting or reversingabnormal protein aggregation comprising contacting in vitro or in vivo acompound of the instant invention with a cell in a therapeuticallyeffective amount to inhibit or reverse abnormal protein aggregation. Incertain embodiments, inhibition of abnormal protein aggregation occursin vivo in a subject with ALS or another neurodegenerative diseasecharacterized by aberrant protein aggregation (e.g., Huntington'sdisease, prion disease, or Alzheimer's disease). In some embodiments,the subject is a mammal. In some embodiments, the subject is a mouse orrat. In some embodiments, the subject is a human.

In certain embodiments, contact occurs in vitro, and the cell is derivedfrom a mammalian cell line. In certain embodiments, contact occurs invitro, and the cell is derived from a PC12 cell line. In certainembodiments, PC12 cells may additionally contain a detectable moiety tomeasure the extent of inhibition of aggregation. In certain embodiments,a detectable moiety is associated with a protein (e.g., SOD1). Incertain embodiments, the detectable moiety is a flourescent moiety(e.g., a YFP tag). In some embodiments, the detectable moiety is aphosphorescent moiety, a radiolabel, or any other detectable moietyknown in the art, and may be detected using any of the methods known inthe art. In some embodiments, the detectable moiety may be detectedusing a high content microscopy system to allow for high-throughputscreening. In certain embodiments, the detectable moiety allows for themeasurement of cell viability.

Assays for the Identification of Compounds that Protect Against ProteinAggregate-Induced Cytotoxicity

The present invention also provides assays for the identification ofcompounds that protect against protein aggregate-induced cytotoxicity.In certain embodiments, the assays are cell protection assays that areused to identify compounds that protect cells from the cytotoxic effectsof aberrant protein aggregation. In some embodiments, the assays areprotein aggregation inhibition assays that are used to identifycompounds that inhibit protein aggregation in a cell or in vitro. Insome embodiments, the inventive assay may screen at least 1,000, 5,000,10,000, 20,000, 30,000, 40,000, or 50,000 compounds in parallel.

Cytotoxicity Protection Assays

Compounds which protect against protein aggregate-induced cytotoxicitycan be identified using methods according to the present invention. Insome embodiments, the present invention provides a method of identifyingcompounds that protect against protein aggregate-induced cytotoxicitycomprising contacting a cell expressing SOD1 or another proteinsusceptible to aggregation with a test compound, incubating the cellwith the test compound under suitable conditions for an amount of timesufficient to observe a protective effect against proteinaggregate-induced cytotoxicity, and then measuring viability in thecells treated with the test compound. In some embodiments, the extent ofprotein aggregation-induced cytotoxicity is measured by determining thelevel of a detectable moiety (e.g., a fluorescent moiety) in the cell.

In certain embodiments, the expressed protein is a mutant SOD1 protein.In some embodiments, the expressed protein is an SOD1 protein associatedwith a detectable moiety. In certain embodiments, the expressed proteinis a fluoresently tagged mutant SOD1 protein, and the flourescent moietyis a YFP tag. In some embodiments, the detectable moiety is aphosphorescent moiety, epitope, or radiolabel. In some embodiments, thedetectable moiety is any suitable detectable moiety known to those orordinary skill in the art and may be detected using any method known inthe art. In some embodiments, the detectable moiety is a fluorescent tag(e.g., a YFP tag) that can be detected with a high content microscopysystem. In some embodiments, the high content microscopy system detectscell viability and facilitates high-throughput screening of a pluralityof compounds.

Cells may be pre-treated with an agent that modulates the expression ofa protein of interest (e.g., SOD1) in the assay. The agent may, forinstance, induce the expression of a gene responsible for the protein ofinterest (e.g., doxycycline-inducible promoter). In some embodiments,cells may also be treated with an agent that modulates proteasomeactivity. In certain embodiments, the agent may be a proteasomeinhibitor (e.g., MG132). In some embodiments, cell viability of cellspre-treated with an agent described herein is measured using methodsdescribed above.

In certain embodiments, the time of incubation of a cell with a testcompound ranges from approximately 1 minute to approximately 1 week. Insome embodiments, the time of incubation ranges from approximately 5minutes to approximately 1 week. In some embodiments, the time ofincubation ranges from approximately 30 minutes to approximately 2 days.In some embodiments, the time of incubation ranges from approximately 30minutes to approximately 1 day. In some embodiments, the time ofincubation ranges from approximately 1 hour to approximately 1 day. Insome embodiments, the time of incubation ranges from approximately 1hour to approximately 18 hours. In some embodiments, the time ofincubation ranges from approximately 1 hour to approximately 12 hours.In some embodiments, the time of incubation ranges from approximately 1hour to approximately 6 hours. In some embodiments, the time ofincubation ranges from approximately 1 hour to approximately 3 hours. Insome embodiments, the time of incubation is approximately 6 hours. Insome embodiments, the time of incubation is approximately 12 hours. Insome embodiments, the time of incubation is approximately 18 hours. Insome embodiments, the time of incubation is approximately 24 hours.

In certain embodiments, the temperature during incubation of a cell witha test compound ranges from approximately 20° C. to approximately 45° C.In certain embodiments, the temperature ranges from approximately 20° C.to approximately 40° C. In certain embodiments, the temperature rangesfrom approximately 25° C. to approximately 40° C. In certainembodiments, the temperature ranges from approximately 30° C. toapproximately 40° C. In certain embodiments, the temperature isapproximately 30° C. In certain embodiments, the temperature isapproximately 37° C.

Compounds that are active in the above-mentioned assay couldtheoretically protect against abnormal protein aggregate-inducedcytotoxicity through a number of biological mechanisms. The presentinvention additionally provides methods to screen for compounds thatprotect against abnormal protein-aggregate induced cytotoxicity whereinthe protein aggregation is inhibited in a non-specific manner.

Compounds which inhibit aberrant protein aggregation can be identifiedusing methods similar to those described above in the aforementionedcytotoxicity assay. In some embodiments, the present invention providesa method of identifying compounds that inhibit aberrant proteinaggregation comprising contacting a cell expressing SOD1 or otherprotein susceptible to aggregation with a test compound, incubating thecell with the test compound under suitable conditions, and thenmeasuring the extent of protein aggregation in the cells treated withthe test compound as compared to a control. In certain embodiments, theextent of inhibition of protein aggregation is measured by staining theprotein aggregates with a detectable stain (e.g., Image-iT plasmamembrane dye). In some embodiments, the detectable stain is detectedusing a scanning device (e.g., Cellomics Arrayscan). In certainembodiments, the protein aggregates are detected using any method ofdetecting protein aggregates known in the art.

Compounds identified using the above-mentioned assays may be furtherexamined using biological assays to guide structure-activityrelationship (SAR) analyses of the identified compounds. Biologicalassays and SAR analyses are known to those of skill in the art.

Method of Protecting Cells from the Cytotoxic Effects of Aggregated SOD1

The present invention provides a method of protecting cells against thecytotoxic effects of aggregated SOD1 protein comprising contacting invitro or in vivo a compound of the invention with a cell in atherapeutically effective amount to protect the cell from the effects ofaggregated SOD1. In certain embodiments, protection of a cell occurs invivo in a subject with ALS. In some embodiments, the subject is amammal. In some embodiments, the subject is a mouse or rat. In someembodiments, the subject is a human.

In other instances, protection of a cell from the cytotoxic effect ofaggregated SOD1 occurs in vitro. In certain embodiments, protectionoccurs in vitro in a cell culture. In some embodiments, compounds of theinvention are contacted with a cell line in vitro and the cell line is amammalian cell line. In certain embodiments, the cell line is the PC12cell line.

In some embodiments, cells are associate with a detectable moiety suchas those described above. In some embodiments, cells contain a proteinlabeled with a detectable moiety. In certain embodiments, the proteinlabeled with a detectable moiety is SOD1 and the detectable moiety is afluoresent moiety. In certain embodiments, the flourescent moiety (e.g.,a YFP tag) that may be detected using a high content microscopy systemto allow for high-throughput screening. In some embodiments, thedetectable moiety is a phosphorescent moiety, an epitope, radiolabel, orany other detectable moiety known in the art, and may be detected usingany of the methods known in the art. In certain embodiments, thedetectable moiety allows for the measurement of cell viability.

Method of Measuring Changes in Ubiquitin Proteasome Activity

The present invention provides a method of measuring changes inubiquitin proteasome activity comprising contacting in vitro or in vivoa compound of the instant invention with a cell in a therapeuticallyeffective amount to effect a change in ubiquitin proteasome activity. Incertain embodiments, modulation of proteasome activity with an inventivecompound occurs in vivo in a subject with ALS. In some embodiments, thesubject is a mammal. In some embodiments, the subject is a mouse or rat.In some embodiments, the subject is a human.

In certain embodiments, contact occurs in vitro by contacting a testcompound with a cell, incubating the test compound with the cell usingmethods described above, and measuring the extent of inhibition ofprotein aggregation in the cell. In certain embodiments, the cell isderived from a mammalian cell line. In some embodiments, the cell isderived from a PC12 cell line or a HeLa cell line. In certainembodiments, the cells contain a detectable moiety to measure the extentto which proteasome activity is inhibited. In some embodiments, cellscontain a protein labeled with a detectable moiety. In certainembodiments, the protein is SOD1 and the detectable moiety is afluoresent moiety. In certain embodiments, the detectable moiety is aflourescent moiety (e.g., a Ubi-YFP tag). In some embodiments, thedetectable moiety is a Ubi-YFP tag, which is detectable by fluorescencemicroscopy. In some embodiments, the detectable moiety is aphosphorescent moiety, an epitope, a radiolabel, or any other detectablemoiety known in the art, and may be detected using any of the methodsknown in the art. In some embodiments, the detectable moiety may bedetected using a high content microscopy system to allow forhigh-throughput screening. In certain embodiments, the detectable moietyallows for the measurement of cell viability.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallycompositions, which comprise a therapeutically effective amount of oneor more of a provided compound, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents for usein treating ALS or any other diseases, disorders, or conditions. Asdescribed in detail, pharmaceutical compositions of the presentinvention may be specially formulated for administration in solid orliquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

Pharmaceutically acceptable salts of provided compounds includeconventional nontoxic salts or quaternary ammonium salts of a compound,e.g., from non-toxic organic or inorganic acids. For example, suchconventional nontoxic salts include those derived from inorganic acidssuch as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric,nitric, and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic,and the like.

In other cases, provided compounds may contain one or more acidicfunctional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. These salts can likewise be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting the purified compound in its free acid form with asuitable base, such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with apharmaceutically-acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. See, for example,Berge et al., supra.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, and the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, this amount will range from about 1% to about 99% of activeingredient, preferably from about 5% to about 70%, most preferably fromabout 10% to about 30%.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a compound of the present invention.

Methods of preparing formulations or compositions comprising providedcompounds include a step of bringing into association a compound of thepresent invention with the carrier and, optionally, one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association a compound of thepresent invention with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol, glycerol monostearate, and non-ionic surfactants;absorbents, such as kaolin and bentonite clay; lubricants, such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, and mixtures thereof; and coloring agents. In the caseof capsules, tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-shelled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

Tablets may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent.

Tablets, and other solid dosage forms of pharmaceutical compositions ofthe present invention, such as dragees, capsules, pills and granules,may optionally be scored or prepared with coatings and shells, such asenteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions that can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of pharmaceutical compositions of the invention for rectalor vaginal administration may be presented as a suppository, which maybe prepared by mixing one or more compounds of the invention with one ormore suitable nonirritating excipients or carriers comprising, forexample, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active compound.

Dosage forms for topical or transdermal administration of a compound ofthis invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Dissolvingor dispersing the compound in the proper medium can make such dosageforms. Absorption enhancers can also be used to increase the flux of thecompound across the skin. Either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel can control therate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

In certain embodiments, a compound or pharmaceutical preparation isadministered orally. In other embodiments, the compound orpharmaceutical preparation is administered intravenously. Alternativerouts of administration include sublingual, intramuscular, andtransdermal administrations.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1% to 99.5% (morepreferably, 0.5% to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In some embodiments, one or more provided compounds, or pharmaceuticalcompositions thereof, is provided to a synucleinopathic subjectchronically. Chronic treatments include any form of repeatedadministration for an extended period of time, such as repeatedadministrations for one or more months, between a month and a year, oneor more years, or longer. In many embodiments, chronic treatmentinvolves administering one or more provided compounds, or pharmaceuticalcompositions thereof, repeatedly over the life of the subject. Preferredchronic treatments involve regular administrations, for example one ormore times a day, one or more times a week, or one or more times amonth. In general, a suitable dose such as a daily dose of one or moreprovided compounds, or pharmaceutical compositions thereof, will be thatamount of the one or more provided compound that is the lowest doseeffective to produce a therapeutic effect. Such an effective dose willgenerally depend upon the factors described above. Generally doses ofthe compounds of this invention for a patient, when used for theindicated effects, will range from about 0.0001 to about 100 mg per kgof body weight per day. Preferably, the daily dosage will range from0.001 to 50 mg of compound per kg of body weight, and even morepreferably from 0.01 to 10 mg of compound per kg of body weight.However, lower or higher doses can be used. In some embodiments, thedose administered to a subject may be modified as the physiology of thesubject changes due to age, disease progression, weight, or otherfactors.

If desired, the effective daily dose of one or more provided compoundsmay be administered as two, three, four, five, six, or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a provided compound to be administered alone,it is preferable to administer a provided compound as a pharmaceuticalformulation (composition) as described above.

Provided compounds may be formulated for administration in anyconvenient way for use in human or veterinary medicine, by analogy withother pharmaceuticals.

According to the invention, provided compounds for treating neurologicalconditions or diseases can be formulated or administered using methodsthat help the compounds cross the blood-brain barrier (BBB). Thevertebrate brain (and CNS) has a unique capillary system unlike that inany other organ in the body. The unique capillary system has morphologiccharacteristics which make up the blood-brain barrier (BBB). Theblood-brain barrier acts as a system-wide cellular membrane thatseparates the brain interstitial space from the blood.

The unique morphologic characteristics of the brain capillaries thatmake up the BBB are: (a) epithelial-like high resistance tight junctionswhich literally cement all endothelia of brain capillaries together, and(b) scanty pinocytosis or transendothelial channels, which are abundantin endothelia of peripheral organs. Due to the unique characteristics ofthe blood-brain barrier, hydrophilic drugs and peptides that readilygain access to other tissues in the body are barred from entry into thebrain or their rates of entry and/or accumulation in the brain are verylow.

In one aspect of the invention, provided compounds that cross the BBBare particularly useful for treating synucleinopathies. In oneembodiment, provided compounds that cross the BBB are particularlyuseful for treating amyotrophic lateral sclerosis (ALS). Therefore itwill be appreciated by a person of ordinary skill in the art that someof the compounds of the invention might readily cross the BBB.Alternatively, the compounds of the invention can be modified, forexample, by the addition of various substitutuents that would make themless hydrophilic and allow them to more readily cross the BBB.

Various strategies have been developed for introducing those drugs intothe brain which otherwise would not cross the blood-brain barrier.Widely used strategies involve invasive procedures where the drug isdelivered directly into the brain. One such procedure is theimplantation of a catheter into the ventricular system to bypass theblood-brain barrier and deliver the drug directly to the brain. Theseprocedures have been used in the treatment of brain diseases which havea predilection for the meninges, e.g., leukemic involvement of the brain(U.S. Pat. No. 4,902,505, incorporated herein in its entirety byreference).

Although invasive procedures for the direct delivery of drugs to thebrain ventricles have experienced some success, they are limited in thatthey may only distribute the drug to superficial areas of the braintissues, and not to the structures deep within the brain. Further, theinvasive procedures are potentially harmful to the patient.

Other approaches to circumventing the blood-brain barrier utilizepharmacologic-based procedures involving drug latentiation or theconversion of hydrophilic drugs into lipid-soluble drugs. The majorityof the latentiation approaches involve blocking the hydroxyl, carboxyland primary amine groups on the drug to make it more lipid-soluble andtherefore more easily able to cross the blood-brain barrier.

Another approach to increasing the permeability of the BBB to drugsinvolves the intra-arterial infusion of hypertonic substances whichtransiently open the blood-brain barrier to allow passage of hydrophilicdrugs. However, hypertonic substances are potentially toxic and maydamage the blood-brain barrier.

Antibodies are another method for delivery of compositions of theinvention. For example, an antibody that is reactive with a transferrinreceptor present on a brain capillary endothelial cell, can beconjugated to a neuropharmaceutical agent to produce anantibody-neuropharmaceutical agent conjugate (U.S. Pat. No. 5,004,697,incorporated herein in its entirety by reference). The method isconducted under conditions whereby the antibody binds to the transferrinreceptor on the brain capillary endothelial cell and theneuropharmaceutical agent is transferred across the blood brain barrierin a pharmaceutically active form. The uptake or transport of antibodiesinto the brain can also be greatly increased by cationizing theantibodies to form cationized antibodies having an isoelectric point ofbetween about 8.0 to 11.0 (U.S. Pat. No. 5,527,527, incorporated hereinin its entirety by reference).

A ligand-neuropharmaceutical agent fusion protein is another methoduseful for delivery of compositions to a host (U.S. Pat. No. 5,977,307,incorporated herein in its entirety by reference). The ligand isreactive with a brain capillary endothelial cell receptor. The method isconducted under conditions whereby the ligand binds to the receptor on abrain capillary endothelial cell and the neuropharmaceutical agent istransferred across the blood brain barrier in a pharmaceutically activeform. In some embodiments, a ligand-neuropharmaceutical agent fusionprotein, which has both ligand binding and neuropharmaceuticalcharacteristics, can be produced as a contiguous protein by usinggenetic engineering techniques. Gene constructs can be preparedcomprising DNA encoding the ligand fused to DNA encoding the protein,polypeptide or peptide to be delivered across the blood brain barrier.The ligand coding sequence and the agent coding sequence are inserted inthe expression vectors in a suitable manner for proper expression of thedesired fusion protein. The gene fusion is expressed as a contiguousprotein molecule containing both a ligand portion and aneuropharmaceutical agent portion.

The permeability of the blood brain barrier can be increased byadministering a blood brain barrier agonist, for example bradykinin(U.S. Pat. No. 5,112,596, incorporated herein in its entirety byreference), or polypeptides called receptor mediated permeabilizers(RMP) (U.S. Pat. No. 5,268,164, incorporated herein in its entirety byreference). Exogenous molecules can be administered to the host'sbloodstream parenterally by subcutaneous, intravenous or intramuscularinjection or by absorption through a bodily tissue, such as thedigestive tract, the respiratory system or the skin. The form in whichthe molecule is administered (e.g., capsule, tablet, solution, emulsion)depends, at least in part, on the route by which it is administered. Theadministration of the exogenous molecule to the host's bloodstream andthe intravenous injection of the agonist of blood-brain barrierpermeability can occur simultaneously or sequentially in time. Forexample, a therapeutic drug can be administered orally in tablet formwhile the intravenous administration of an agonist of blood-brainbarrier permeability is given later (e.g., between 30 minutes later andseveral hours later). This allows time for the drug to be absorbed inthe gastrointestinal tract and taken up by the bloodstream before theagonist is given to increase the permeability of the blood-brain barrierto the drug. On the other hand, an agonist of blood-brain barrierpermeability (e.g., bradykinin) can be administered before or at thesame time as an intravenous injection of a drug. Thus, the term“co-administration” is used herein to mean that the agonist ofblood-brain barrier and the exogenous molecule will be administered attimes that will achieve significant concentrations in the blood forproducing the simultaneous effects of increasing the permeability of theblood-brain barrier and allowing the maximum passage of the exogenousmolecule from the blood to the cells of the central nervous system.

In other embodiments, a provided compound can be formulated as a prodrugwith a fatty acid carrier (and optionally with another neuroactivedrug). The prodrug is stable in the environment of both the stomach andthe bloodstream and may be delivered by ingestion. The prodrug passesreadily through the blood brain barrier. The prodrug preferably has abrain penetration index of at least two times the brain penetrationindex of the drug alone. Once in the central nervous system, theprodrug, which preferably is inactive, is hydrolyzed into the fatty acidcarrier and a provided compound or analog thereof (and optionallyanother drug). The carrier preferably is a normal component of thecentral nervous system and is inactive and harmless. The compound and/ordrug, once released from the fatty acid carrier, is active. Preferably,the fatty acid carrier is a partially-saturated straight chain moleculehaving between about 16 and 26 carbon atoms, and more preferably 20 and24 carbon atoms. Examples of fatty acid carriers are provided in U.S.Pat. Nos. 4,939,174; 4,933,324; 5,994,932; 6,107,499; 6,258,836; and6,407,137, the disclosures of which are incorporated herein by referencein their entirety.

Administration of agents of the present invention may be for eitherprophylactic or therapeutic purposes. When provided prophylactically,the agent is provided in advance of disease symptoms. The prophylacticadministration of the agent serves to prevent or reduce the rate ofonset of symptoms of ALS. When provided therapeutically, the agent isprovided at (or shortly after) the onset of the appearance of symptomsof actual disease. In some embodiments, the therapeutic administrationof the agent serves to reduce the severity and duration of the disease.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples describedbelow. The following examples are intended to illustrate the benefits ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLES Example 1 High Throughput Assays for Protection from MutantSOD1-Induced Cytotoxicity

Cultured cells are utilized to conduct high throughput assays forcompounds that protect against mutant SOD1-induced cytotoxicity. Twoassays are used: first, in the cytotoxicity protection assay, compoundsare screened for their ability to protect cells from the cytotoxiceffects of aggregated mutant SOD1, irrespective of mechanism of drugaction. Second, in the protein aggregation assay, compounds are screenedfor their ability to reduce aggregation of mutant SOD1. The highthroughput cytotoxicity protection assay is the primary screen andcompounds active in the primary screen (and their analogs) move forwardinto the secondary screen for protein aggregation.

Assay for Protection Against Mutant SOD1-Induced Cytotoxicity

The high throughput cytotoxicity protection assay was carried out inPC12 cells that express mutant G93A SOD1 as a YFP fusion protein from adoxycycline-inducible promoter. Several lines of evidence suggest thatcytotoxicity of protein aggregates in ALS is due at least in part toinhibition of the proteasome (Bruijin et al. Annu. Rev. Neurosci. 2004,27, 723-729; Cleveland et al. Nat. Rev. Neurosci. 2001, 2(11), 806).This idea was tested by examining the sensitivity of PC12 cells to SOD1aggregates in the presence and absence of proteasome inhibitor MG132.PC12 cells expressing no SOD1, wild type SOD1, G85R SOD1 or G93A SOD1were grown with or without MG132 (FIG. 1). Cells expressing no SOD1,wild type SOD1 and G85R SOD1 were relatively insensitive to MG132, withan IC_(so) of approximately 400 nM. In contrast, cells expressing G93ASOD1 were approximately 5-fold more sensitive to MG132 (IC₅₀ ˜75 nM). Inthese cells, protein aggregation was detected after 24 h and loss ofcell viability was detected at approximately 48 h. Qualitatively similarresults were obtained with the structurally distinct proteasomeinhibitor bortezomib (Velcade®), suggesting that PC12 cells are indeedsusceptible to proteasome inhibition and not some other effect of MG132.The ability of protein aggregates to induce cell death was examined bytreating G93A SOD1-expressing cells with MG132 for 24 h, removing theMG132 by washing and assaying cell viability after another 24 h. Becausethe loss of cell viability was similar following MG132 removal (FIG. 1,part C), it is likely that mutant SOD1 aggregates contribute directly tocytotoxicity in PC12 cells. However, this effect is specific for G93ASOD1 suggesting that this mutant may produce higher levels of a toxicaggregated form of SOD1.

Based on these results, a high throughput screen was developed forcompounds that protect against the cytotoxicity of G93A SOD1 proteinaggregates using geldanamycin or radicicol as a positive control. PC12cells expressing G93A SOD1 were treated with 100 nM MG132 with orwithout co-treatment with geldanamycin or radicicol. The lattercompounds inhibit the chaperone HSP90 and induce expression of otherchaperones. As anticipated, radicicol reduced formation of proteinaggregates and increased cell viability in a dose-dependent manner (FIG.2). Statistical analysis of the data produced a Z′ value of 0.55, whichwould predict good performance as a positive control in a highthroughput screen (Zhang et al., J. Biomol. Screen 1999, 4(2), 67-73).

Mutant SOD1 Direct Protein Aggregation Assay.

Compounds that are active in the above assay could theoretically protectagainst mutant SOD1-induced cytotoxicity through a number of mechanisms,including the following: 1) compounds could nonspecifically block orreverse protein aggregation via chaperone induction, as observed forradicicol and geldanamycin; 2) compounds could block or reverse theaggregation of a specific aggregated protein form; 3) compounds couldinterfere with an event downstream of protein aggregation that plays acritical role in mutant SOD1-induced cytotoxicity (e.g., proteasomefunction); and 4) compounds could act directly on SOD1 in a manner thatprevents mutant SOD1 aggregation. These possibilities were tested usingan assay that directly measures protein aggregation. In addition, unlikethe high throughput cytotoxicity protection assay, the proteinaggregation assay is based on G85R SOD1; this broadens the scope of thescreening strategy, and should eliminate compounds with highly specificactivity (i.e., G93A SOD 1 limited) against protein aggregation.

In PC12 cells that express wild-type SOD1, SOD1 was diffusely localizedthroughout the cell (Matsumoto et al., J. Cell. Biol. 2005, 171(1),75-85). In contrast, G85R SOD1 showed heterogeneous patterns oflocalization; in most cells, G85R was diffusely localized throughout thecell, but in −5% of the cells, G85R SOD1 was localized in largeperi-nuclear aggregates. In cells treated with MG132, up to 75% of cellsexpressing G85R SOD1 contain such protein aggregates (FIG. 3), but noaggregation was observed in cells expressing wild-type SOD1. Cellsexpressing G93A mutant SOD1 showed an intermediate level of proteinaggregation: none of the cells developed protein aggregates in theabsence of MG132, and ˜75% of the cells had protein aggregates followingtreatment with MG132 (FIG. 3). Similar effects were observed in cellstreated with bortezomib (Velcade®). Therefore, these effects are likelyto be due to MG132-induced proteasome inhibition, and not due to anoff-target effect of MG132.

The sensitivity of this assay was optimized by selecting conditions thatmaximize the difference between active and inactive samples. Theidentification of a positive control is a crucial step in assaydevelopment. Thus, PC12 cells expressing G85R or G93A mutant SOD1 weretreated with MG132 to induce protein aggregation, and then co-treatedwith candidate chemical suppressors of protein aggregation. Twocompounds with similar activity were identified in these experiments:geldanamycin and radicicol. Both compounds induce heat shocktranscription factor NSF-1, which in turn induces the heat shockresponse (FIG. 4). Treatment with radicicol reduced the proportion ofcells with aggregates from 75% to 25%, a sufficient difference to allowvisual scoring for compounds with efficacy equal to or greater thanradicicol.

To allow this assay to be used in a high-throughput manner, a CellomicsArrayscan® high content microscopy system was used for screening andquantification. Initial experiments indicated that G85R SOD1 aggregateswere more readily recognized by the high content microscopy system andits computer algorithm. Because the most robust high content assaysmeasure events on a per cell basis, it was necessary to select afluorescent stain that marks whole cells to be used with a compatiblestain that marks intracellular structures. On the basis of pilotexperiments with a number of vital dyes, an Image-iT conjugated wheatgerm agglutinin (WGA) dye from Molecular Probes was selected and acomputer algorithm for detecting WGA was developed. As shown in FIG. 5,WGA provided an excellent cellular marker that did not interfere withdetection of YFP-tagged SOD1.

Initial experiments used geldanamycin and radicicol as positivecontrols, both of which are reproducibly active, but did not show highlystatistically reliable performance when the high content microscopysystem was used. Therefore, pyrazolones CMB-3350 and CMB-3319 weretested as alternate positive controls. CMB-3350 and CMB-3319 wereselected because of their outstanding activity in the SOD1 cytotoxicityprotection assay.

Example 2 Procedures and Protocols for High Throughput Screening MutantSOD1-Induced Cytotoxicity Protection Assay: Automated High ThroughputScreening Protocol.

PC12 cells were maintained in Dulbecco's Modified Eagle's Medium(Invitrogen), supplemented with 10% horse serum, 0.1 μ/ml NGF (Sigma),and 0.1 μ/ml doxycycline (Sigma). Cells were incubated at 37° C. in ahumidified atmosphere with 5% CO₂. The expression of SOD1 protein wasinduced by the withdrawal of doxycycline from the media. For screeningin microwell format, cells were seeded at 15,000 cells/well in 96-wellplates (Falcon-BD) using a Multidrop 384 (Thermo Lab Systems) andincubated 24 h prior to compound addition. Compounds (control and test)were added to wells using a Zymark Sciclone. MG132 was added 24 h laterusing the Multidrop 384. Cell viability was measured after 48 h usingthe fluorescent viability probe calcein-AM (Molecular Probes).Fluorescence intensity was measured using a POLARstar fluorescence platereader (BMG). All experiments included positive control wells treatedwith radicicol at 700 nM and negative control wells treated with DMSO.Fluorescence data were coupled with compound structural data, thenstored and analyzed using the Cambridgesoft Chemoffice Enterprise Ultrasoftware package.

Mutant SOD1 Aggregation Assay: Automated High Content ScreeningProtocol.

PC12 cells were maintained in Dulbecco's Modified Eagle's Medium(Invitrogen) supplemented with 10% horse serum, plus 0.1 μ/ml NGF(Sigma) and 0.1 μg/ml doxycycline (Sigma). Cells were incubated at 37°C. in a humidified atmosphere with 5% CO₂ and are routinely subculturedusing 0.05% trypsin-EDTA solution. The expression of SOD1 protein wasinduced by the withdrawal of doxycycline from the media. For screeningin microwell format, cells were seeded at 5,000 cells/well in 96-wellplates (Falcon-BD) using a Thermo Labsystems Multidrop 384. Compounds(control and test) were added to wells using a Zymark Sciclone. Testcompounds were incubated with the cells for 24 h prior to the additionof MG132. CMB-3350 was used as a positive control at 5 μM, DMSO as anegative control and test compounds were applied at 512M. MG132 was thenadded at 1 μM final concentration and the cells were grown for anadditional 24 hours. To assay protein aggregation, cell culture mediumwas then removed and the cells were washed once with HBSS then incubatedin HBSS containing 5 μg/ml of the Image-iT WGA plasma membrane dye(Molecular Probes) for 15 min at 37° C. Cells were then washed with PBSand imaged using the Cellomics Arrayscan®.

Assay Automation.

Arrayscan images were recorded using a 20× objective and the XF93-TRITCfilter set in channel 1 for the Image-iT WGA plasma membrane dye or theXF93-FITC filter set for SOD1-YFP and analyzed using Cellomics spotdetector software. Software parameters were set to maximallydifferentiate cells from background and maximize recognition of SOD1aggregates without recognizing background cytoplasmic staining as aspot. Data were recorded as spot count (aggregate)/object (cell).

A library was screened using the cytotoxicity protection assay as theprimary screen and the protein aggregation assay as the secondaryscreen. The overall screen produced an average Z′ factor of 0.5 and anaverage Z factor of 0.6. Using 60% viability as the cutoff for activity,195 primary actives were recovered (0.38% primary hit rate). All primaryactives were retested in dose response curves yielding 68 confirmedactives (0.13% confirmed hit rate). These actives were then tested forauto-fluorescence and inhibition of protein synthesis, potential causesof artifactual positives. Confirmed primary actives were assayed in thehigh content protein aggregation screen and grouped into compounds thatwere positive or negative in the secondary screen. Cheminformaticanalysis of these compounds identified 17 chemotypes ofstructurally-related active and inactive compounds plus 15 singletonhits. Most active compounds were also active in the secondary highcontent assay, two of the confirmed primary actives were not. Initialbiological analysis focused on the arylsulfanyl pyrazolone lead series.The compound collection includes riluzole, which tested negativeindicating that the actives tested here differ from the sole clinicallyapproved drug for ALS.

Counterscreens.

All active compounds were tested for autofluorescence, a potentialartifact in any fluorescence assay. This test only identified onecompound with significant autofluorescence. This low rate ofautofluorescence might be due to the use of calcein-AM to measure cellviability. Calcein-AM is a high quantum yield reagent and thus onlyhighly fluorescent compounds produce a comparable signal. All activecompounds were tested for ability to inhibit protein synthesis, as thiscould artificially reduce protein aggregation. Specific reduction in thecellular concentration of SOD1 was estimated using a modification of thehigh content assay, in which YFP fluorescence was measured with a platereader prior to counting the number of cells per well with ArrayScan.This yielded a value for YFP fluorescence per cell, which is equivalentto SOD1 content per cell. No compounds tested active in this assay.Lastly, all active compounds were tested for nonspecific cytotoxicity inuntransfected PC12 cells. The vast majority of the active compoundslacked or had extremely low non-specific cytotoxicity. One possibleexplanation for the low level of cytotoxicity associated with activecompounds is that the primary protection screen selected specificallyagainst such compounds.

Discovery and Preliminary SAR of Chemical Lead Series.

Results show that certain provided compounds and analogs thereof wereactive (ED₅₀≦20 μM) in the cytotoxicity protection assay. Compounds thatwere tested in the SOD1 protein aggregation assay are also indicated.

Chemical Identification and Analog Selection.

All active compounds were reordered and re-tested from dry powder toconfirm activity. Confirmed active compounds were characterized by aniterative process designed to establish preliminary SAR. This processincluded reanalyzing the screening files to identifystructurally-related but weakly active or inactive compounds and testingstructurally-related compounds purchased from commercial suppliers. Over300 chemical compounds were acquired from an extensive search of over 2million unique available commercial compounds on the basis of asubstructure and Tanimoto similarity analysis carried out using anin-house vendor chemical warehouse database system. All compounds were≧90% pure (by LC/MS and/or ¹H NMR).

Biological Assays to Guide SAR Analysis.

The high throughput cytotoxicity protection assay and the high contentprotein aggregation assay only score compounds as active or inactive.Two quantitative metrics were therefore developed to evaluate compoundactivity for SAR development: potency (ED₅₀), which is the concentrationproducing half maximal cell viability, and efficacy, which is defined asthe maximum viability produced by a compound at its optimum dosage.Cytotoxicity is measured by determining the concentration that reducesPC12 cell viability by 50% and is reported as the cytotoxicity IC₅₀(FIG. 2). Compounds were also tested for nonspecific inhibition ofprotein synthesis, inhibition of protein aggregation (i.e., SOD1-YFPfusion) and autofluorescence (to ensure the detection method was notdetecting false positives).

Summary of Biological Effects.

Most of the active compounds showed 100% efficacy as compared with thepositive control, radicicol, which showed only 80% efficacy at the mostefficacious dose. The most potent compounds produce ED₅₀ values between100 nM and 2 μM in the cytotoxicity protection assay. Compounds thatwere active in the cytotoxicity protection assay were active in theprotein aggregation assay with comparable potency (Table 2). Thus, it isreasonable to assume that these compounds reduce cytotoxicity byreducing mutant SOD1 aggregation. These compounds are generally notcytotoxic or only very weakly cytotoxic. Structure-activity relationshipstudies of provided compounds were conducted by comparing the activityof compounds with substitutions at N-2, C-4 and C-5 around the pyrazolering. Compounds without substitution at N-2 (R1) or N-2 (R1) & C-4 (R3)that contain an arylsulfanyl moiety at C-5 (R2) were most potent, withED50 values between 0.374 and 5 μM. Alkene substituted compounds at C-4,such as dimethyl amine and various substituted aromatic and heterocyclicgroups, with a C-5 arylsulfanyl group are equally potent, with ED₅₀values between 0.6 and 5 μM. All substituted alkenes were tested asmixed isomers (E and Z). Table 3 lists selected compounds in this serieswith an ED₅₀<μM in the cytotoxicity protection assay. The most potentactive compounds (CMB-3319 & CMB-3350) are the C-5 substituted pyrazolescontaining an arylsulfanyl group. CMB-3299 is similar to CMB-3319, buthas a methyl substitution at C2 rather than a chlorine group on thearylsulfanyl ring system.

Mechanistic Studies on Arylsulfanyl Pyrazolones.

Mechanism of action studies have been initiated for provided compounds.provided compounds active in the cytotoxicity protection assay wereinitially assayed for the ability to prevent SOD1 aggregation usingmanual, low-throughput methods. The results were consistent with theautomated screening results, indicating that all provided compoundsreduced aggregation of G93A SOD1 in PC12 cells with similar efficacy andpotency. The ability of provided compounds to induce expression ofmolecular chaperones was also tested using stably-transfected HeLa cells(HeLa hse-luc) expressing a luciferase reporter gene under the controlof the human HSP70 promoter. This HeLa hse-luc reporter system was usedpreviously to identify small-molecule activators of the heat shockresponse (Westerheide et al. J. Biol. Chem. 2004, 279(53), 56053-56060).The results showed that the five most potent provided compounds, at upto 100 μM, failed to induce luciferase activity in the HeLa hse-lucsystem, while positive control compounds celastrol and CdCl₂ stronglyinduced luciferase activity (FIG. 6). Provided compounds also failed toinduce expression of multiple Hsp70, Hsp40, and Hsp90 genes asdetermined by RT-PCR. These results suggest that while providedcompounds are likely to protect PC12 cells from G93A SOD1 by reducingits aggregation, this effect is not likely to be mediated by increasingthe expression of molecular chaperones.

Since the CMB-003299 cytotoxicity of SOD1 protein aggregates may involveinhibition of proteasome activity, compounds that stimulate proteasomeactivity could reverse or prevent mutant SOD1-induced cytotoxicity.Thus, active compounds were tested for the ability to stimulateproteasome activity using a cell line expressing ubiquitin-conjugatedYFP [Ubi-YFP 74]. In these cells, increased proteasome activity causesincreased degradation of Ubi-YFP and decreased YFP fluorescence.Conversely, compounds that inhibit proteasome activity increaseaccumulation of Ubi-YFP and enhanced YFP fluorescence. As expected,MG132 augments YFP fluorescence intensity ˜3-fold compared to untreatedcontrols (FIG. 7). The provided compounds (CMB-3299 or CMB-3319)strongly inhibit MG132-induced Ubi-YFP fluorescence, suggesting thatthese compounds stimulate proteasome activity. To demonstrate that thesecompounds are not simply blocking the action of MG132, a group ofprovided compounds were tested on a C. elegans strain which expressesmutant SOD1 in muscle cells. Protein aggregates are present in theabsence of MG132 and aggregation is suppressed following treatment witha provided compound. These results suggest that the mechanism by whichprovided compounds protect cells against mutant SOD1-inducedcytotoxicity is by stimulating the proteasome.

Example 3 Procedures and Protocols for Compound CharacterizationActivation of Cellular Heat Shock Response.

HeLa cells, stably transfected with a luciferase reporter plasmid undercontrol of the HSP70 promoter, were plated in a 96 well plate at adensity of 7500 cells/100 μl. Following a 16 h incubation at 37° C./5%CO₂, compounds were added and cells incubated for an additional 8 h.Cells were lysed by the addition of 100 μl BrightGlo reagent (Promega)according to manufacturer's instructions and luciferase activitymeasured by quantifying luminescence signal. Cells were treated withtest compounds at concentrations ranging from 10-100 μM celastrol orCdCl₂ serve as positive controls while DMSO is a negative control.

Proteasome Activity Assay.

HeLa cells (20,000/chamber) were plated on 8 chambered Tissue-tekcoverglass pretreated with 1 mg/ml poly-D-lysine and allowed to adherefor 18 h at 37° C./5% CO₂. Cells were transfected with 0.2 μg eachCMV-CFP and Ubi-YFP plasmids using Lipofectamine 2000 according tomanufacturer's instructions. Cells were allowed to express CFP andUbi-YFP for 18 h followed by a 6 h incubation with test compounds (25μM). MG132 (1 μM) was added for a final 18 h incubation and Ubi-YFPdegradation assessed by fluorescence microscopy. Fluorescence intensityof CFP and YFP was calculated on a cell-by-cell basis from capturedimages using AxioVision software (Zeiss). YFP fluorescence wasnormalized to CFP fluorescence.

Cytotoxicity Assay.

Nonspecific cytotoxicity was determined using PC12 and other cell linesin conventional dose response assays employing calcein-AM fluorescenceas the indicator of cell viability. In addition, compounds could score afalse positive by selectively reducing the synthesis of aggregatedproteins (i.e., SOD1-YFP fusion). Reduction in SOD1 proteinconcentration was determined using a modification of the high contentassay in which YFP fluorescence is measured on a plate reader prior todetermining number of cells per well with the Arrayscan. Specific YFPactivity, which is equivalent to SOD1 content per cell, was thencalculated.

Expanded Assays for Aggregated Protein Spectrum.

Provided compounds are tested for efficacy in cell lines that expresshuntingtin protein aggregates instead of SOD1 (Kim et al., Nat. Cell.Biol. 2002, 4(10), 826-831; Matsumoto et al., J. Biol. Chem. 2005).Other active compound series are tested in these cells as well. Ifbroader characterization is desired, analogous cell lines expressingtau, amyloid Aβ, and prion protein are constructed and similarexperiments performed. In addition, a cell line that expresses TDP-43and produces TDP-43 aggregates is constructed. TDP-43 is an aggregatedprotein that accumulates in neural cells in patients with sporadic ALS(Neumann et al. Science, 2006, 314(5796), 130-133; Arai et al., Biochem.Biophys. Res. Commun. 2006, 351(3), 602-611). Active compounds aretested for ability to prevent TDP-43 aggregation and/or reduce itsassociated cytotoxicity. Compounds that prevent TDP-43-associatedcytotoxicity and/or aggregation are expected to have greater potentialfor efficacy in sporadic ALS patients.

Identification of Chemical Lead(s) Whose Predicted PharmacologicalProperties are Suitable for Testing in Mice.

The most common and most rigorous approach to determine the toxicity andpharmacological properties of candidate pharmacological compounds relieson in vivo testing in laboratory animals. Because animal testing is bothexpensive and time consuming, many drug discovery organizations haveturned to in vitro methods to analyze the pharmacological properties ofcompounds during structural optimization. This approach relies onminiaturized predictive in vitro ADMET assays that are amenable to high-to medium-throughput implementation. These methods have achievedincreased popularity, both because animal models are limited in theirability to predict efficacy and toxicity in humans, and becauseregulatory agencies have begun to require human in vitro testing, suchas human liver CYP450 inhibition assays, prior to human clinical trials.The need for methods of this type has also been motivated by the switchfrom animal disease models to target based in vitro methods for leaddiscovery (Kerns et al. Curr. Top Med. Chem. 2002, 2(1), 87-98; Di etal., Curr. Opin. Chem. Biol. 2003, 7(3) 402-408; Kassel et al., Curr.Opin. Chem. Biol. 2004, 8(3), 339-345). The in vitro ADMET approach isbased on the use of a suite of chemical tests (compound integrity,compound solubility, compound aggregation, lipophilicity, pKa) andbiological assays (Caco-2 and/or PAMPA assays, cytochrome P450metabolism and inhibition, cardiac risk, and cytotoxicity) that assessthe absorption, distribution, metabolism, excretion, and toxic effectsof test compounds. The output from ADMET assays is used to identify andselect for compounds with low predicted toxicity and desirable predictedpharmacological properties during SAR-based optimization. Compoundsoptimized by this approach are selected for analysis in the mouse modelof ALS. The following suite of assays are performed:

Cytotoxicity: Cytotoxicity is determined in cultured cells.

Compound Purity: Each compound is subjected to chemical analysis toconfirm molecular weight and determine purity. Only compounds >95% pureare used for further testing. Compounds <80% pure are re-purified andre-tested.

Compound Aggregation: Aggregation of screening hits is measured usingdynamic light scattering.

Solubility: Because compounds are stored in DMSO, it is necessary todetermine aqueous solubility of initial hit compounds and their analogs.High solubility in aqueous solution is necessary for high GI absorption,bioavailability, and for chemical formulation. Compounds should besoluble in aqueous solution at >10 μg/ml. Solubility of >50 μg/ml ispreferred.

General Permeability: High membrane permeability is required foreffective GI absorption and optimal bioavailability. Passivepermeability is typically assessed in the PAMPA artificial membraneassay (Kansy et al., J. Med. Chem. 2002, 45(8), 1712-1722). Cell-basedpermeability determinations using the Caco-2 cell assay are moreresource intensive but more predictive of active transport or efflux invivo (Artursson et al., Adv. Drug. Deliv. Rev. 2001, 46(1-3), 27-43).Caco-2 assays are performed as needed during SAR development.

Blood Brain Barrier: CNS therapeutics must penetrate the blood-brainbarrier (BBB) to achieve in vivo efficacy (Basak et al., Pharm Res.1996, 13(5), 775-778). A QSAR model was developed for predicting in vivoBBB partitioning using the logarithm of the blood-brain concentrationratio as a diagnostic indicator. A 189 compound dataset was constructedfrom data in the literature (Rose et al., J. Chem. Inf. Comp. Sci. 2002,42(3), 651-656; Pan et al., J. Chem. Inf. Comp. Sci. 2004, 44(6),2083-2098) and compounds in the dataset entered as 2D structuraldrawings with ISIS/Draw and saved as mol files and converted into 3Dstructures using the Corina software prior to calculating moleculardescriptors using Dragon. A Support Vector Machine (SVM) linearregression algorithm was used to generate & validate the model. Theprediction accuracy (Q) for the SVM linear regression training model(n=166) and validation set (n=24) was 86.75 and 86.96%, respectively,confirming the validity of the model. Using this BBB predictive model,the potential of active compounds to cross the BBB is appraised earlyduring development. Other BBB predictive models, including a variant ofthe PAMPA assay developed specifically for this purpose (Di et al., Eur.J. Med. Chem. 2003, 38(3), 223-232), is used as needed. Results of theseexperiments guide synthesis of compounds with appropriate ADMEproperties for use as CNS therapeutics.

Lipophilicity and pKa: Lipophilicity is determined via octanol-waterpartition at pH 7.4 (Hitzel et al., Pharm. Res. 2000, 17(11), 1389-1395)and pKa is determined by capillary electrophoresis and photodiode arraydetection (Ishihama et al., J. Pharm. Sci. 2002. 91(4), 933-942).

Metabolism (microsome, S9 fraction, CYP450): Liver Cytochrome P450(CYP450) enzymes are the major route for xenobiotic metabolism andmicrosomal and hepatocyte stability is the best predictor ofpharmacokinetic half-life. Cross-species comparisons of metabolism inliver microsomes can predict potential issues with liver toxicity inhumans. Selected active compounds are tested in liver microsomes fromefficacy species (mouse), toxicity species (rat, dog, monkey), andhumans for metabolic stability.

Drug-drug interaction and cardiac risk potential: Data-mining algorithmsand data compiled from the literature, are used to predict whetheractive compounds are likely to interact with and/or inhibit majorCytochrome P450's (1A2, 2C9. 2C19, 3A4, 2D6) (Kerns et al., Curr. Top.Med. Chem. 2002, 2(1), 87-98). Synthesized compounds are also testeddirectly for ability to bind and inhibit human CYP P450. If potentialproblems are indicated by these approaches, analog synthesis is directedtowards developing alternative compounds. This approach allows for theassessment of cardiac safety, since hERG inhibition is assessedsimultaneously on the same compounds. The hERG assay is done with thefast-patch methodology. hERG ion channel inhibition is implicated ingreater than 90% of reported cases of cardiac toxicity, and is a commoncause of after market drug failures and withdrawals. Information on hERGinhibition as well as inhibition of five major human CYP450s (1A2, 2C9.2C19, 3A4, 2D6) allows for accurate prediction of potentialdrug-associated cardiac risk.

Plasma stability: Plasma stability of active compounds is assessed inefficacy species (mouse), toxicity species (rat, dog, monkey), and humancells.

Plasma protein binding: Plasma protein binding is examined in efficacyspecies (mouse), toxicity species (rat, dog, monkey), and human cellsusing ultrafiltration methodology.

The compounds identified were evaluated in the transgenic G93A ALS mousemodel. ADME data was used to determine optimal route of administration.LD₅₀ and maximally-tolerated dose were determined, and that informationwas used to design and execute pharmacokinetic studies to assess brainbioavailability, and to guide choice of dosing regimen (i.e., frequency,dose, route). Three-dose basic efficacy studies were performed oncompounds demonstrating acceptable tolerability and bioavailability. Forcompounds that demonstrate efficacy, a more complete preclinical studyis performed and the efficacies of the test compound and previouslycharacterized neuroprotective agents are compared. This may includefurther dose optimization, phenotype assessment, comparison withcompounds of established efficacy, and analysis of brain tissue intreated animals.

Procedures

Subjects. In the present study, G93A SOD1 mice and littermate controlswere bred from existing colonies at the Bedford VA Medical Hospital. Themale G93A SOD1 mice were mated with B6SJL females and the offspring weregenotyped by PCR using tail DNA. The number of SOD1 transgenes wereassessed periodically by PCR to ensure that transgene copy number didnot increase or decrease significantly over the course of time. Micewere housed in micro-isolator cages in complete barrier facilities. A 12hour light-dark cycle was maintained and animals were given free accessto food and water. Control and transgenic mice of the same age (±2 days)and from the same ‘f’ generation were selected from multiple litters toform experimental cohorts (n=20 per group). Standardized criteria forage and parentage were used for placing individual mice intoexperimental groups/cohorts. Wild type mice were used for initialtoxicity, tolerability, and pharmacokinetic studies and ALS mice wereused for one month tolerability studies.

Tolerability, Dosing, and Pharmacokinetics. The tolerable dose range andLD50 for each test compound was determined in wild type mice byincreasing the dose b.i.d. one-fold each injection. The route ofadministration (p.o. by gavage or i.p.) and starting dose was based onsolubility and other output from ADME studies. Initial pharmacokinetic(pK) studies were conducted by giving animals a single dose, sacrificingthem after 30 min, 1 h, 2 h, 4 h, 6 h, or 12 h, and dissecting brainsand spinal cords and determining drug concentration in the targettissue. Drug steady-state levels were determined in animals that havebeen dosed for 1 week prior to sacrifice. These data are used tooptimize the dosing regimen for efficacy studies. Drug doses shouldachieve a desirable drug concentration in the brain and spinal cord oftreated mice. Working doses at least 10-fold lower than the acutetolerability dose are preferable.

Efficacy studies. Efficacy is measured using endpoints that indicateneuroprotective function. These include reversal of degenerative lesionsin the brain and neuronal tissues, improved motor function, increasedbody weight, and prolonged survival. Some mice cohorts are sacrificed ata predetermined time point, while others are sacrificed when they reachcriteria for euthanasia.

Survival. Mice were observed three times daily (morning, noon, and lateafternoon) throughout the experiment. Mice were euthanized when diseaseprogression is sufficiently severe that they were unable to initiatemovement and right themselves after gentle prodding for 30 seconds.

Body weights. Mice are weighed twice a week at the same time each day.Weight loss is a sensitive measure of disease progression in transgenicG93A SOD1 mice and of toxicity in transgenic and wild type mice.

Motor/behavioral. Quantitative methods of testing motor function areused including Rotarod and analysis of open field behavior. Decline ofmotor function is a sensitive measure of disease onset and progression.

Neuropathology. Selected cohorts (n=10) of treated and untreated G93ASOD1 mice are euthanized at 120 days for isolation and analysis ofspinal cord tissue. For this purpose, mice are deeply anesthetized andperfused transcardially with 4% buffered paraformaldehyde at the desiredtime point. These and other studies are performed in a blinded manner,to avoid bias in interpretation of the results. Brains are weighed,serially sectioned at 50 μm and stained for quantitative morphology(cresyl violet), to assess protein aggregates, and to label ventralneurons. Remaining tissue samples/sections are stored for future use.Stereology is used to quantify ventral horn atrophy, neuronal atrophy,neuronal loss, and protein aggregate load.

Analysis. Data sets are generated and analyzed for each clinical andneuropathological measure. Effects on behavior and neuropathology arecompared in treatment and control groups. Dose-dependent effects areassessed in each treatment group using multiple two-sided ANOVA tests.Multiple comparisons in the same subject groups are dealt with post hoc.Kaplan-Meier analysis was used for survival and behavioral function.

Neuronal quantitation. Serial lumbar spinal cord tissue sections (n=20)from L3-L5 spinal cord segments are used for gross spinal cord areas andneuronal analysis. Gross areas of the spinal cord sections arequantified from each experimental cohort using NIH Image. From the samesections, the ventral horn is delineated by a line from the centralcanal laterally and circumscribing the belly of gray matter. Absoluteneuronal counts of Nissl-positive neurons are performed in the ventralhorns in the lumbar spinal cord. Only those neurons with nuclei arecounted. All counts are performed with the experimenter (JM) blinded totreatment conditions. Counts are performed using Image J (NIH) andmanually verified and the data represent the average neuronal numberfrom the sections used.

Interpretation. Compound efficacy is evaluated using behavioral andneuropathological endpoints. Results for the test compound are comparedwith results from compounds with established efficacy andneuroprotective action in the G93A SOD1 mouse model. These experimentsdirectly test whether the provided compounds provide therapeutic benefitand, if so, the magnitude of the benefit. Along the way, usefulinformation about solubility, administration, and toxicity are alsoobtained.

Example 4 Synthetic Routes

Synthetic schemes of the selected analogs of the generalarylsulfanylpyrazolone scaffold are illustrated below.

^(a)Reagents and conditions: (a) benzaldehyde, HCl, rt, 5 min; (b)NH₂NH₂, EtOH, reflux, 1 h; (c) PBr₃, THF, rt, 6 h; (d) benzenethiol,K₂CO₃, DMF, 70° C., 18 h.

3-Benzylidenefuran-2,4(3H,5H)-dione (TC-I-49): Tetronic acid (300 mg, 3mmol) was added to benzaldehyde (1.0 mL, 9 mmol), and the resultingsolution was stirred. HCl (37.7%, 0.1 mL) was added drop wise. Thereaction mixture was vigorously stirred until it solidified. The solidproduct was crushed, soaked in hexane and washed with hexane. The crudeproduct was purified by recrystallization (ethyl acetate/hexanes=8/2) togive compound TC-I-49 (0.27 g, 48%) as a yellow solid.

4-Benzylidene-3-(hydroxymethyl)-1H-pyrazol-5(4H)-one (TC-I-53): To asuspension of ethanol (2 mL) and TC-I-49 (367 mg, 2 mmol) was added anethanolic solution of hydrazine (2 N, 1 mL). The resulting suspensionwas refluxed for 1 h, during which time all of the TC-I-49 dissolved anda new precipitate formed. The resulting mixture was cooled in anice-bath for 20 min. The white precipitate was filtered and washed withcold ethanol to give TC-I-53 (77 mg, 28%).

4-Benzylidene-3-(bromomethyl)-1H-pyrazol-5(4H)-one (TC-I-82): PBr₃ (30μl, 0.38 mmol) was added to a suspension of TC-I-53 (0.23 g, 1.1 mmol)in THF (5 mL) at 0° C. under N₂. The reaction mixture was warmed to roomtemperature after 5 min. Additional PBr₃ (30 μl, 0.38 mmol) was addedafter 1 h. The resulting suspension gradually became a solution and wasstirred overnight. The reaction mixture was then quenched with brine,and the aqueous layer was extracted with CH₂Cl₂. The combined organiclayer was washed with brine, dried over Na₂SO₄, and evaporated todryness. TC-I-82 (0.125 g, 41%) was obtained by flash columnchromatography (ethyl acetate/hexanes=1/4) as a yellow solid.

4-Benzylidene-3-(phenylthiomethyl)-1H-pyrazol-5(4H)-one (TC-I-91):Benzenethiol (61 μL, 0.60 mmol) was added to a suspension of TC-I-82(0.13 g, 0.50 mmol) and anhydrous potassium carbonate (0.42 g, 3.0 mmol)in DMF (2 ml) at room temperature. The reaction temperature was gentlybrought to 70° C. After the resulting suspension was stirred for 18 h,the reaction mixture was quenched with HCl (0.25 N), and the aqueouslayer was extracted with EtOAc. The combined organic layer was washedwith brine, dried over Na₂SO₄, and evaporated to dryness. TC-I-91 (40mg, 27%) was obtained by flash column chromatography (ethylacetate/hexanes=1/2) as a white solid.

^(a)Reagents and conditions: (a) ethyl 4-chloroacetoacetate, Et₃N, 0°C., 30 min; (b) NH₂NH₂, EtOH, reflux, overnight.

Ethyl 4-(4-chlorophenylthio)-3-oxobutanoate (TC-I-164):4-Chlorothiophenol (1.1 g, 7.61 mmol) was mixed with ethyl4-chloro-acetoacetate (0.95 mL, 7.00 mmol) in CH₂Cl₂ (100 mL) at 0° C.Triethyl amine (1.5 mL, 10.8 mmol) was then added drop wise. After theresulting suspension was stirred at 0° C. for another 30 min, thereaction mixture was poured into water, and the aqueous layer wasextracted with EtOAc. The combined organic layer was washed by saturatedNaHCO₃, HCl (0.25 N), brine, concentrated in vacuo, and purified byflash column chromatography (ethyl acetate/hexanes=1/9) to affordTC-1-164 (1.82 g, 96%) as light yellow oil. Exposure to hydrazine inrefluxing ethanol, generated TC-II-165.

5-((4-Chlorophenylthio)methyl)-1H-pyrazol-3(2H)-one (TC-I-165): CompoundTC-I-164 (0.48 g, 1.76 mmol) was stirred in EtOH (5 mL), and anethanolic solution of NH₂NH₂ (2 N, 0.88 mL, 1.76 mmol) was added. Theresulting solution was refluxed for overnight under Ar, during which aprecipitate formed. The reaction mixture was then cooled at roomtemperature. The precipitate was washed with cold EtOH and dried invacuo to afford TC-I-165 (0.31 g, 72%) as a white solid.

Reagents and conditions: (a) TBHP, vanadyl acetylacetonate, CH₂Cl₂, roomtemp, overnight; (b) NH₂NH₂, EtOH, room temp, overnight.

Ethyl 4-(4-chlorophenylsulfinyl)-3-oxobutanoate (TC-II-65): CompoundTC-I-164 (1.27 g, 4.66 mmol) was mixed with t-butyl hydrogen peroxide(70 wt % in water, 1.1 mL, 7.69 mmol) in CH₂Cl₂ (50 mL) at roomtemperature, and vanadyl acetylacetonate (0.1% mol) was added slowly.Extra t-butyl hydrogen peroxide (0.5 mL, 3.50 mmol) was added to thereaction mixture after 2 h. The resulting suspension was stirredovernight at room temperature. The reaction mixture was thenconcentrated in vacuum and purified by flash column chromatography(ethyl acetate/hexanes=1/4) to afford compound TC-II-65 (0.98 g, 73%) asa light yellow oil. Exposure to hydrazine in refluxing ethanol,generated TC-II-68.

Reagents and conditions: (a) TBHP, vanadyl acetylacetonate, CH₂Cl₂, roomtemp, overnight; (b) NH₂NH₂, EtOH, room temp, overnight.

Ethyl 4-(4-chlorophenylsulfonyl)-3-oxobutanoate (TC-II-64): CompoundTC-I-164 (3.00 g, 11.0 mmol) was mixed with t-butyl hydrogen peroxide(70 wt % in water, 1.5 mL, 10.50 mmol) in CH₂Cl₂ (50 mL) at roomtemperature, and vanadyl acetylacetonate (0.1% mol) was added slowly.Extra t-butyl hydrogen peroxide (6 mL, 41.94 mmol) was added to thereaction mixture after 2 h. The resulting suspension was stirredovernight at room temperature. The reaction mixture was thenconcentrated in vacuum and purified by flash column chromatography(ethyl acetate/hexanes=1/4) to afford compound TC-II-64 (1.61 g, 48%) asa light yellow oil. Exposure to hydrazine in refluxing ethanol,generated TC-II-70.

Reagents and conditions: (a) ethyl 4-chloroacetoacetate, Et₃N, 0° C., 30min; (b) H₂O₂, AcOH, EtOAc, room temp, overnight; (c) NH₂NH₂, EtOH, roomtemp, overnight.

Ethyl 4-(4-chloro-2,5-dimethylphenylsulfonyl)-3-oxobutanoate(TC-II-171): Compound TC-II-170 (4.68 g, 14.1 mmol) was mixed with AcOH(5 mL) in EtOAc (10 mL), and H₂O₂ (30% in water, 10 mL, 84.6 mmol) wasadded. The resulting solution was left stirring at room temperature forovernight after extra H₂O₂ (30% in water, 5 mL, 42.3 mmol) was added.The reaction mixture was then evaporated in vacuo and purified by flashcolumn chromatography (ethyl acetate/hexanes=1/3) to afford TC-II-171(4.34 g, 85%) as a light yellow oil. Exposure to hydrazine in refluxingethanol, generated TC-II-172.

Reagents and conditions: (a) ethyl 4-chloroacetoacetate, NaH, dry THF,dry DMF, 70° C., overnight; (b) NH₂NH₂, EtOH, room temp, overnight.

Ethyl 4-(4-chlorophenoxy)-3-oxobutanoate (TC-II-48): A solution of4-chlorophenol (6.4 g, 50 mmol) in THF (25 mL) was treated with NaH (60%in mineral oil, 2 g, 50 mmol) at 0° C. In another flask, a solution ofethyl 4-chloroacetoacetate (10.21 mL, 75 mmol) in THF (25 mL) wastreated NaH (60% in mineral oil, 3.5 g, 75 mmol) at −20° C. Theresulting yellowish suspension was slowly added to the solution ofsodium 4-chlorophenoxide, which was kept at 0° C. After the addition ofDMF (10 mL), the reaction temperature was slowly raised to 70° C. Afterreaction mixture was stirred at 70° C. for overnight, it was cooled andevaporated to dryness. The residue was purified by flash columnchromatography (ethyl acetate/hexanes=1/9) to afford TC-II-148 as ayellowish oil, which still contained some 4-chloroacetoacetate. Nofurther purification was applied for the next step of synthesis.Exposure to hydrazine in refluxing ethanol, generated TC-II-150.

Reagents and conditions: (a) EtOAc, LDA, THF, −78° C. to rt, overnight;(b) NH₂NH₂, EtOH, rt, overnight.

Reagents and conditions: (a) NH₂NHMe, EtOH, room temp, overnight; (b)Me₂SO₄, CaO, MeOH, room temp, overnight

5-((4-Chlorophenoxy)methyl)-1,2-dimethyl-1H-pyrazol-3(2H)-one(TC-III-93): Compound TC-III-87 (0.145 g, 0.61 mmol) was mixed with CaO(0.20 g, 3.57 mmol) and Me₂SO₄ (0.3 mL, 1.80 mmol) in MeOH (10 mL). Theresulting suspension was left stirring at room temperature forovernight. The reaction mixture was then evaporated to dryness. Theresidue was purified by flash column chromatography (ethylacetate/hexanes=2/1) to afford TC-III-93 (28.5 mg, 19%) as a whitesolid.

Reagents and conditions: (a) EtOAc, LDA, THF, −78° C. to rt, overnight;(b) NH₂NH₂, EtOH, rt, overnight.

4-Chlorophenyl ethyl malonate (TC-III-185): EtOAc (0.63 mL. 6.44 mmol)was added to a solution of n-BuLi (1.6 M in hexanes, 9.2 mL, 14.72 mmol)and diisopropylamine (2.1 mL, 14.85 mmol) at 0° C. After 60 min ofstirring, a THF solution of the acetyl chloride (1.31 g, 6.41 mmol) wasadded at −78° C. The reaction mixture was stirred at −78° C. for 1 h,then at room temperature for overnight. The resulting reaction solutionwas quenched with diluted HCl (0.25 M), and the aqueous layer wasextracted with Et₂O. The combined organic layer was dried over Na₂SO₄and concentrated in vacuo. The residue was purified by flash columnchromatography (ethyl acetate/hexanes=1/9) to give TC-III-85 (0.41 g,25%) as a yellowish oil.

^(a)Reagents and conditions: (a1) NaOEt, EtOH, 70° C., overnight; (a2)DIEA, DCM, rt, 30 min; (b) EtOAc, LiHMDS, THF, −78° C., 8 h; (c) NH₂NH₂,EtOH, rt, overnight.

2-(3,5-Dichlorophenoxy)-N-methoxy-N-methylacetamide (TC-IV-10), methodal: To a solution of phenol (5.27 g, 32.33 mmol) in EtOH (10 mL) wasadded NaOEt (21 wt % in EtOH, 12.1 mL, 32.41 mmol) at room temperature.The reaction mixture was stirred for 10 min.2-Bromo-N-methoxy-N-methylacetamide (5.87 g, 32.25 mmol) was then gentlyadded at room temperature. After the resulting solution was stirred at70° C. for overnight, the reaction mixture was cooled, poured into HCl(0.25 M), and the aqueous layer was extracted with EtOAc. The combinedorganic layer was concentrated in vacuo and reconstituted in CHCl₃. Theprecipitate was filtered and washed with CHCl₃. Wienreb amide TC-IV-10(4.53 g, 53%) was obtained as a white solid.

2-(3,5-Dichlorophenoxy)-N-methoxy-N-methylacetamide (TC-IV-10), methoda2: To a solution of N,O-dimethylhydroxylamine HCl salt (5.3 g, 54.33mmol) in DCM (250 mL) was added DIEA (24 mL, 137.8 mmol) at roomtemperature. The reaction mixture was stirred for 5 min. Acetyl chloride(13.0 g, 54.28 mmol) was then added in DCM drop by drop at 0° C. Thereaction mixture was stirred at room temperature for another 30 min. Theresulting reaction solution was washed with HCl (1 N) and concentratedunder vacuum. The crude solid product was washed with Et₂O to giveWeinreb amide TC-IV-10 (12 g, 84%) as a white solid.

Ethyl 4-(3,5-dichlorophenoxy)-3-oxobutanoate (TC-IV-12): EtOAc (0.9 mL.9.19 mmol) was added to a solution of LiHMDS (1 N in THF, 21 mL, 21mmol) at 0° C. and stirred. After 60 min, a THF solution of the Wienrebamide TC-IV-10 (2.4 g, 9.19 mmol) was added at −78° C. After theresulting solution was stirred at −78° C. for 8 h, the reaction mixturewas quenched with diluted HCl (0.25 N), and the aqueous layer wasextracted with Et₂O. The combined organic layer was dried over Na₂SO₄and concentrated in vacuo. The residue was purified by flash columnchromatography (ethyl acetate/hexanes=1/9) to give TC-IV-12 (1.03 g,39%) as a white solid.

5-((3,5-Dichlorophenoxy)methyl)-1H-pyrazol-3(2H)-one (TC-IV-19):Ethanolic hydrazine (2 N, 2.5 mL, 5 mmol) was added to a solution ofTC-IV-12 (1.5 g, 5.15 mmol) in EtOH (25 mL). The resulting solution wasstirred at room temperature for overnight. The reaction mixture was thenevaporated in vacuo, purified by flash column chromatography (ethylacetate/hexanes=1/2) and recrystallized in ethyl acetate/hexanes to givethe final product TC-IV-19 (0.368 g, 28%) as a white crystal.

Reagents and conditions: (a) PdCl₂(PPh₃), K₂CO₃, dioxane, H₂O, 100° C.,16 h; (b) NaOEt, EtOH, 70° C., overnight; (c) EtOAc, LiHMDS, THF −78°C., overnight; (d) NH₂NH₂, EtOH, rt, overnight.

Biphenyl-3-ol (TC-IV-119): 3-Bromophenol (1.0 g, 5.78 mmol),phenylboronic acid (1.4 g, 11.48 mmol), potassium carbonate (2.0 g,14.47 mmol), and PdCl₂(PPh₃) (1/200 eq.) were added to a solution ofdioxane/H₂O (20 mL/5 mL). The resulting solution was refluxed for 16 h.The reaction mixture was then partitioned between Et₂O and water, andthe aqueous phase was extracted with Et₂O. The combined organic layerwas evaporated to dryness and purified by flash column chromatography(ethyl acetate/hexanes=1/9) to give TC-IV-119 (1.02 g, 100%) as atransparent oil.

TC-IV-120: 3,5-Dibromophenol (1.0 g, 3.97 mmol), phenylboronic acid (2g, 16.4 mmol), potassium carbonate (2.7 g, 19.5 mmol), and PdCl₂(PPh₃)(1/200 eq.) were added to a solution of dioxane/H₂O (20 mL/5 mL). Theresulting solution was refluxed for 16 h. The reaction mixture was thenpartitioned between Et₂O and water, and the aqueous phase was extractedwith Et₂O. The combined organic layer was evaporated to dryness andpurified by flash column chromatography (ethyl acetate/hexanes=1/9) togive TC-IV-120 (0.83 g, 83%) as a transparent oil.

Reagents and conditions: (a) DIEA, DCM, rt, 48 h; (b) K₂CO₃, acentone,50° C., overnight; (c) EtOAc, LiHMDS, THF −78° C., 16 h; (d) NH₂NH₂,EtOH, rt, overnight; (e) HBr, PhOH, H₂O, 100° C., 8 h; (f) R_(A)CHO,NaBH(OAc)₃, DCM, rt, overnight.

N-(3,5-Dichlorophenyl)benzenesulfonamide (TC-IV-145): Benzenesulfonylchloride (1.74 mL, 13.57 mmol) and DIEA (0.34 mL, 2.49 mmol) were addedto a solution of 3,5-dichloroaniline (2.0 g, 12.34 mmol) in DCM (50 mL).The resulting solution was stirred at room temperature for 48 h, duringwhich a precipitate was formed. The reaction mixture was then evaporatedto dryness and purified by flash column chromatography (ethylacetate/hexanes=1/9) to give TC-IV-145 (1.90 g, 51%) as a yellow solid.

2-(N-(3,5-Dichlorophenyl)phenylsulfonamido)-N-methoxy-N-methylacetamide(TC-IV-146): To a solution of TC-IV-145 (1.90 g, 6.29 mmol) in acetone(30 mL) was added 2-bromo-N-methoxy-N-methylacetamide (1.14 g, 6.26mmol) and potassium carbonate (0.86 g, 6.22 mmol) at room temperature.After the resulting suspension was stirred at 50° C. for overnight, thereaction mixture was concentrated in vacuo and purified by flash columnchromatography (ethyl acetate/hexanes=1/4) to give the Wienreb amideTC-IV-146 (1.15 g, 44%) as a yellow solid.

Ethyl 4-(N-(3,5-dichlorophenyl)phenylsulfonamido)-3-oxobutanoate(TC-IV-147): EtOAc (0.28 mL. 2.86 mmol) was added to a solution ofLiHMDS (1 N in THF, 6.3 mL, 6.3 mmol) at −78° C. and stirred. After 60min, a THF solution of the Weinreb amide TC-IV-146 (1.15 g, 2.86 mmol)was added. After the resulting solution was stirred at −78° C. forovernight, the reaction mixture was quenched with diluted HCl (0.25 N),and the aqueous layer was extracted with Et₂O. The combined organiclayer was dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by flash column chromatography (ethyl acetate/hexanes=1/2) togive crude TC-IV-147 (0.60 g, 48%) as a yellow oil.

N-(3,5-Dichlorophenyl)-N-((5-oxo-2,5-dihydro-1H-pyrazol-3-yl)methyl)benzenesulfonamide(TC-IV-150): Ethanolic hydrazine (2 N, 0.69 mL, 1.38 mmol) was added toa solution of TC-IV-147 (0.60 g, 1.39 mmol) in EtOH (10 mL). After theresulting solution was stirred at room temperature for overnight, thereaction mixture was evaporated in vacuo and purified by flash columnchromatography (ethyl acetate/hexanes=1/1) to give TC-IV-150 (0.20 g,32%) as a white solid.

5-((3,5-Dichlorophenylamino)methyl)-1H-pyrazol-3(2H)-one (TC-IV-154):TC-IV-150 (30 mg, 0.068 mmol) and phenol (60 mg, 0.64 mmol) were addedto a solution of HBr in water (48%, 0.75 mL). After the resultingsuspension was stirred at 100° C. for 8 h, the reaction mixture waspartitioned between water and EtOAc, and the aqueous layer was extractedwith EtOAc. The combined organic layer was evaporated to dryness andpurified by flash column chromatography (ethyl acetate) to giveTC-IV-154 (10 mg, 57%) as a white solid.

Reagents and conditions: (a) K₂CO₃, n-BuOH, reflux, 72 h; (b) K₂CO₃,acentone, 50° C., overnight; (c) EtOAc, LiHMDS, THF −78° C., 16 h; (d)NH₂NH₂, EtOH, rt, overnight.

1-(3,5-Difluorophenyl)piperazine (TC-IV-174): Bis(2-chloroethyl)aminehydrochloride (2.07 g, 11.60 mmol) was added to a solution of3,5-difluoroaniline (1.50 g, 11.62 mmol) in n-BuOH (20 mL). After theresulting solution was refluxed for 48 h, anhydrous potassium carbonate(1.61 g, 11.62 mmol) was added. After being refluxed for another 24 h,the reaction mixture was cooled, partitioned between water and CHCl₃,and the aqueous layer was extracted with CHCl₃. The combined organiclayer was washed with water, dried over Na₂SO₄, and evaporated todryness. Et₂O and 1 N HCl/EtOH were added to the residue, and theprecipitate was filtered to give TC-IV-174 (1.17 g, 30%) as a whitesolid.

Reagents and conditions: (a) NiCl₂(dppp), THF, 60° C., 6 h; (b) (1)BBr₃, DCM, −78° C. to rt, 1 h; (2) H₂O, −78° C. to rt, 1 h.

Scheme 14 illustrates the use of Kumada couplings to access synthonswherein R⁴ is cyclic aliphatic.

Reagents and conditions: (a) K₂CO₃, n-BuOH, reflux, 48° C.; (b) K₂CO₃,acetone, 50° C., overnight; (c) EtOAc, LiHMDS, THF, −78° C., 16 h; (d)NH₂NH₂, EtOH, rt, overnight.

TABLE 2 ¹H NMR data for selected compounds. Structure ¹H NMR

¹H NMR (500 MHz, DMSO-d₆, δ): 11.62 (br s, 1H), 9.72 (br s, 1H), 7.57(s, 1H), 7.46 (s, 1H), 5.39 (s, 1H), 4.15 (s, 2H), 2.30 (s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.29 (s, 1H), 8.03 (s, 1H), 7.68-7.18 (m,10H), 5.19 (dd, J = 32.0, 16.5 Hz, 2H)

¹NMR (500 MHz, acetone-d₆, δ): 10.89 (s, 1H), 8.03 (s, 1H), 7.73 (d, J =6.50, 2H), 7.78-7.22 (m, 7H), 5.22 (dd, J = 16.0, 22.0 Hz)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.54 (br s, 1H), 9.55 (br s, 1H),7.34-7.29 (m, 4H), 7.19 (t, J = 7.0 Hz, 1H), 5.31 (s, 1H), 4.07 (s, 2H)

1H NMR (500 MHz, DMSO-d₆, δ): 11.22 (br s, 1H), 10.07 (br s, 1H),7.33-7.26 (m, 4H), 5.34 (s, 1H), 4.04 (s, 2H), 1.25 (s, 9H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11. 64 (br s, 1H), 9.52 (br s, 1H), 8.13(d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 5.41 (s, 1H), 4.26 (s,2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.55 (br s, 1H), 9.47 (br s, 1H), 7.36(s, 4H), 5.31 (s, 1H), 4.08 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.45 (br s, 1H), 9.79 (br s, 1H), 7.48(d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 5.32 (s, 1H), 4.08 (s,2H)

¹H NMR (400 MHz, DMSO-d₆, δ): 10.66 (br s, 1H), 7.59 (s, 1H), 7.52 (d, J= 8.4 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 5.34 (s, 1H), 4.14 (s, 2H)

¹H NMR (400 MHz, DMSO-d₆, δ): 9.92 (br s, 1H), 7.63 (s, 1H), 7.47-7.39(m, 2H), 5.38 (s, 1H), 4.15 (s, 2H)

¹H NMR (400 MHz, DMSO-d₆, δ): 7.20 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8Hz, 2H), 5.20 (s, 1H). 3.83 (s, 2H), 2.88 (s, 6H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.67 (br s, 1H), 7.67 (d, J = 8.5 Hz,2H), 7.45-7.36 (m, 6H), 7.24 (t, J = 7.5 Hz, 1H), 5.46 (s, 1H), 4.11 (s,2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.52 (br s, 1H), 9.40 (br s, 1H),7.40-7.37 (m, 2H), 7.17 (t, J = 8.8 Hz, 2H), 5.27 (s, 1H), 4.03 (s, 1H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.55 (br s, 1H), 9.38 (br s, 1H), 7.16(s, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 7.0 Hz, 1H), 5.33 (s,1H), 4.03 (s, 2H), 2.25 (s, 3H), 2.19 (s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.45 (br s, 1H), 9.36 (br s, 1H), 7.29(d, J = 8.5 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 5.22 (s, 1H), 3.93 (s,2H), 3.73 (s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.49 (br s, 1H), 9.75 (br s, 1H), 7.62(d, J = 8.0 Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H), 5.17 (s, 1H), 4.13-3.99(m, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.24 (br s, 1H), 7.75 (d, J = 9.0 Hz,2H), 7.67 (d, J = 8.5 Hz, 2H), 5.21 (s, 1H), 4.56 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.45 (br s, 1H), 9.35 (br s, 1H), 7.55(d, J = 8.5 Hz, 2H), 7.38 (t, J = 8.0 Hz, 1H), 5.12 (s, 1H), 4.01 (s,2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.56(br s, 1H), 9.47 (br s, 1H), 7.32 (s,1H). 7.26 (s, 1H), 5.33 (s, 1H), 4.05 (s, 2H), 2.27 (s, 3H), 2.19 (s,3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.52 (br s, 1H), 9.50 (br s, 1H), 7.58(s, 1H), 7.37 (s, 1H), 5.22 (s, 1H), 4.03- 3.93 (m, 2H), 2.35 (s, 3H),2.15 (s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.57 (br s, 1H), 9.41 (br s, 1H), 7.50(m, 2H), 7.25-7.21 (m, 1H), 5.35 (s, 1H), 4.12 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.60 (br s, 1H), 9.54 (br s, 1H),7.46-7.17 (m, 4H), 5.39 (s, 1H), 4.14 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.60 (br s, 1H), 9.54 (br s, 1H),7.40-7.22 (m, 4H), 5.33 (s, 1H), 4.13 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.57 (br s, 1H), 9.44 (br s, 1H), 7.39(s, 3H), 5.34 (s, 1H), 4.18 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.42 (br s, 1H), 9.58 (br s, 1H), 7.35(dd, J = 28.0, 8.5 Hz, 4H, 5.35 (s, 1H), 3.70 (s, 2H), 3.49 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.48 (br s, 1H), 9.48 (br s, 1H), 7.36(s, 4H), 5.30 (s, 1H), 4.08 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 10.88 (br s, 1H), 7.35 (s, 4H), 5.24 (s,1H), 3.99 (s, 2H), 3.43 (s, 3H)

¹H NMR (500 MHz, CDCl₃, δ ): 3.88 (s, 2H), 3.32 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.39 (br s, 1H), 9.52 (br s, 1H),7.66-7.34 (m, 9H), 5.36 (s, 1H), 4.12 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.53 (br s, 1H), 9.37 (br s, 1H), 7.37(d, J = 8.0 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.20-7.14 (m, 2H), 5.29(s, 1H), 4.02 (s, 2H), 1.15 (d, J = 6.5 Hz, 6H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.50 (br s, 1H), 9.40 (br s, 1H), 7.28(t, J = 7.8 Hz, 2H), 6.94-6.90 (m, 3H), 5.39 (s, 1H), 4.08 (t, J = 6.8Hz, 2H), 3.69 (s, 2H), 2.82 (t, J = 6.5 Hz, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.54 (br s, 1H), 9.36 (br s, 1H),7.36-7.14 (m, 4H), 5.31 (s, 1H), 4.04 (s, 2H), 2.63 (q, J = 7.4 Hz, 2H),1.12 (t, J = 7.5 Hz, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.79 (br s, 1H), 9.56 (br s, 1H), 7.33(d, J = 9.0 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 5.52 (s, 1H), 4.92 (s,2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.37 (br s, 1H), 9.72 (br s, 1H), 7.61(d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 4.48 (s, 2H), 3.29 (s,1H), 2.71 (q, J = 7.5 Hz, 2H), 1.21 (t, J = 7.5 Hz, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.56 (br s, 1H), 9.48 (br s, 1H), 7.64(d, J = 7.5 Hz, 2H), 7.44 (m, 2H), 5.18 (s, 1H), 4.52 (s, 2H), 2.67 (t,J = 7.5 hz, 2H), 1.60-1.54 (m, 2H), 1.33-1.26 (m, 2H), 0.89 (t, J = 7.3Hz, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.62 (br s, 1H), 9.59 (br s, 1H), 7.67(s, 1H), 7.54 (s, 1H), 5.24 (s, 1H), 4.49 (s, 2H), 2.47 (s, 3H), 2.33(s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.79 (br s, 1H), 9.55 (br s, 1H), 7.18(t, J = 7.8 Hz, 1H), 6.83-6.79 (m, 3H), 5.52 (s, 1H), 4.89 (s, 2H), 2.56(q, J = 7.5 Hz, 2H), 1.16 (t, J = 7.5 Hz, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.72 (br s, 1H), 9.50 (br s, 1H), 7.20(t, J = 8.0 Hz, 1H), 6.97-6.80 (m, 3H), 5.52 (s, 1H), 4.91 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.75 (br s, 1H), 9.47 (br s, 1H), 7.17(t, J = 7.5 Hz, 1H), 6.80-6.75 (m, 3H), 5.54 (s, 1H), 4.90 (s, 2H), 1.54(m, 2H), 1.26-1.23 (m, 26H), 0.85 (t, J = 5.8 Hz, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.82 (br s, 1H), 9.54 (br s, 1H), 7.16(s, 1H), 7.12 (d, J = 1.5 Hz, 2H), 5.53 (s, 1H), 4.99 (s, 2H)

. ¹H NMR (500 MHz, DMSO-d₆, δ): 11.62 (br s, 1H), 9.55 (br s, 1H),7.77-7.36 (m, 2H), 5.26 (s, 1H), 4.70 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.75 (br s, 1H), 9.50 (br s, 1H), 7.10(d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 5.50 (s, 1H), 4.87 (s,2H), 2.54-2.51 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.79 (br s, 1H), 9.51 (br s, 1H), 7.52(d, J = 9.5 Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 7.02 (dd, J = 9.0, 2.5Hz, 1H), 5.56 (s, 1H), 4.98 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.62 (br s, 1H), 9.50 (br s, 1H), 7.99(s, 2H), 7.86 (s, 1H), 5.35 (s, 1H), 4.31 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 10.95 (s, 1H), 7.13 (s, 1H), 7.10 (s, 2H),5.41 (s, 1H), 4.88 (s, 2H), 3.49 (s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 10.98 (br s, 1H), 7.30 (d, J = 8.5 Hz,2H), 7.00 (d, J = 8.5 Hz, 2H), 5.40 (s, 1H), 4.82 (s, 2H), 3.48 (s, 3H)

¹H NMR (500 MHz, CDCl₃, δ ): 7.22-7.19 (m, 2H), 6.93- 6.90 (m, 2H), 5.60(s, 1H), 4.91 (s, 2H), 3.86 (s, 3H), 3.61 (s, 3H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.82 (br s, 1H), 9.53 (br s, 1H),6.80-6.79 (m, 3H), 5.56 (s, 1H), 4.95 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.80 (br s, 1H), 9.52 (br s, 1H), 7.39(s, 1H), 7.28 (m, 2H), 5.55 (s, 1H), 4.99 (s, 1H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.78 (br s, 1H), 9.50 (br s, 1H),7.26-7.23 (m, 2H), 7.14-7.13 (m, 1H), 7.02-7.00 (m, 2H), 5.58 (s, 1H),4.96 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.80 (br s, 1H), 9.50 (br s, 1H),7.67-6.99 (m, 9H), 5.55 (s, 1H), 5.02 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.82 (br s, 1H), 9.50 (br s, 1H), 7.76(d, J = 8.0 Hz, 4H), 7.50-7.38 (m, 7H), 7.27 (s, 2H), 5.60 (s, 1H), 5.13(s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.56 (br s, 1H), 9.39 (br s, 1H),7.76-7.55 (m, 6H), 7.14 (s, 2H), 5.22 (s, 1H), 4.67 (s, 2H)

¹H NMR (500 MHz, DMSO-d₆, δ): 11.56 (br s, 1H), 9.52 (br s, 1H),6.64-6.59 (m, 3H), 5.35 (s, 1H), 4.10 (d, J = 5.0 Hz, 2H)

Example 6 Biological Activity of Synthesized ArylsulfanylpyrazoloneAnalogs

Compounds synthesized above underwent a mutant SOD1 proteinaggregation-induced toxicity protection screen and a direct proteinaggregation high content assay. Results differ within a reasonable rangefrom week to week due to bio-variation, however, relative potencies ofthe analogs were reproducible.

TABLE 3 Best Corresponding Corresponding EC50 (uM) after Entry StructureEC50 (μM) 3299 EC50 (μM) % viability Normalization 1

0.4 2

>32 3

6.85 0.27 77 10.1 4

5.38 0.93 150 2.31 5

5.59 0.93 136 2.4 6

4.49 0.93 93 1.93 7

4.24 0.93 94 1.8 8

2.6 0.55 113 1.89 9

1.58 0.55 124 1.15 10

1.65 77 11

5.11 0.59 109 3.46 12

4.73 0.55 104 3.44 13

0.22 77 14

4.21 0.93 102 1.81 15

>32 16

6.92 0.59 110 4.7 17

0.26 0.11 142 0.95 18

0.43 0.44 140 0.39 19

4.47 0.62 123 2.88 20

1.61 68 21

0.84 0.11 123 3.05 22

0.25 0.09 148 1.11 23

0.08 0.19 134 0.17 24

0.3 0.41 129 0.29 25

4.14 75 26

7.97 0.59 121 5.4 27

9.44 0.93 115 4.06 28

0.4 0.33 106 0.48 29

6.21 0.33 97 7.52 30

2.06 0.33 106 2.5 31

0.57 0.35 131 0.65 32

0.69 0.35 117 0.79 33

3.43 1.79 120 0.77 34

0.99 0.44 101 0.9 35

0.75 0.44 103 0.68 36

0.99 0.44 93 0.9 37

2.28 0.62 116 1.47 38

1.12 0.62 125 0.72 39

1.06 0.49 141 0.87 40

2.08 0.37 66 2.24 41

0.05 0.3 129 0.067 42

0.55 0.72 123 0.31 43

0.36 0.19 130 0.76 44

0.67 0.19 132 1.41 45

3.29 0.67 88 1.96 46

0.13 0.09 136 0.58 47

0.13 0.09 120 0.58 48

6.67 0.52 109 5.13 49

7.6 0.52 107 5.85 50

3.21 1.2 125 1.07 51

1.54 1.2 125 0.51 52

3.06 1.2 144 1.02 53

8.53 1.2 96 2.84 54

11.08 1.2 68 3.7 55

9.04 2.52 111 1.43 56

2.11 2.52 128 0.33 57

6.2 58

4.2 59

>32

In vitro ADME study of CMB-003319. CMB-003319 was evaluated for its invitro ADME properties. The study showed that CMB-003319 has a mediummetabolic potential, medium CaCO-2 permeability, poor plasma stability,and medium aqueous-solubility.

Rat liver microsomal stability study of TC-I-165. TC-I-165 was appliedfor an NADPH dependent rat liver microsomal stability study by HPLC.Preliminary results showed that TC-I-165 has poor microsomal stabilitylike CMB-003319. The test (TC-I-165), positive control (minaprine), andinternal standard (haloperidol) are shown in FIG. 11.

Microsomal metabolite study of TC-I-165. TC-I-165 was used in a NADPHdependent rat liver microsomal metabolite study by HPLC. TC-II-68 andTC-II-70 were proposed as possible metabolites and prepared before thestudy. Results showed that only TC-II-68 was a direct metabolite.

Microsomal metabolite study of TC-I-165. TC-II-125 was prepared as adouble ¹⁵N isotope labeled TC-I-165. TC-I-165, TC-II-125, and TC-II-68were sent for NADPH dependent rat liver microsomal metabolitedetermination. The results showed that during the first hour the onlydirect metabolite from TC-I-165 was TC-II-68, with no nitrogen loss onthe pyrazolone ring.

Rat liver microsomal stability of TC-II-70. TC-II-70 was applied for anNADPH dependent rat liver microsomal stability test by HPLC. The resultsshowed that TC-II-70 is much more stable compared with TC-I-165 for thefirst hour of microsomal metabolism.

In vitro ADME properties of analogs. Compounds were sent for in vitroADME tests to evaluate their ADME properties. By substituting thethioether linkage with an ether linkage or a sulfone linkage in thearylsulfanylpyrazolone scaffold, the microsomal stability andaqueous-solubility were largely improved.

In vitro ADME study of CMB-087229. CMB-087229 was sent for an evaluationof its in vitro ADME properties. The study showed that CMB-087229 has apotency (ED₅₀) of 74 nM, medium human metabolic potential (T_(1/2)=93minutes), medium mouse metabolic potential (T_(1/2)=36 minutes), highpredicted CaCO-2 permeability (A-B=37×10⁻⁶ cm/s), medium plasmastability (T_(1/2)=17 minutes), and aqueous solubility of 250 μM.CMB-087229 has a metabolic half-life in rats of approximately 2 hours.

UV Spectra and Chromatography

Using a UV/VIS spectrometer (Perkin Elmer, Lamda 10), samples werescanned from 200-500 nm to determine the maximum UV absorbance. Awavelength of 260 nm was found to be optimal for HPLC monitoring.Samples were analyzed with a Beckman HPLC using a 166 UV detectorcoupled to a reverse phase C18 analytical column (Phenomenex, Luna 5μC18 (2), 250*4.60 mm). After separation of the samples on the C18 columnusing an acetonitrile-water HPLC program, peaks were analyzed by 32Karat™ software, version 5.1.

Microsomal Stability Study

Test agents (15 μM) were incubated in Eppendorf tubes with mousemicrosomes (pooled male rat liver microsomes (Sprague Dawley), BDScience) at 37° C. Each reaction mixture was carried out in PBS (pH7.4), which contained 1 mg/mL microsomal protein with 1.3 mM NADP⁺, 3.3mM glucose-6-phosphate, 0.4 U/mt glucose-6-phosphate dehydrogenase, and3.3 mM magnesium chloride. A control using minaprine was run to test theactivity of the microsomal activity. After 0 and 20 minutes incubation,40 μL of acetonitrile was added to quench the reactions. The reactionmixture was vortexed and incubated in an ice bath for 2 h. Haloperidol(100 μL, 100 μM) was added to every test tube as an internal standard.The samples were diluted in water and centrifuged to remove theprecipitated protein. The supernatant solutions were loaded for solidphase extraction. Extraction fractions were evaporated and reconstitutedfor HPLC analysis.

Excel was used for data processing. The Response Ratio (RR) wascalculated by dividing the peak area of the analyte by the peak area ofthe internal standard. Data suggest that TC-I-165 has low NADPHdependent rat liver microsomal stability.

TABLE 4 Microsomal stability data processing Sample Name RR Mean % Mean± S.E. Mina 0 min 22.88  100 ± 7.11 Mina 20 min 13.54 59.2 ± 14.9TC-I-165 0 min 2.529  100 ± 26.4 TC-I-165 20 min 0.314 12.4 ± 0.89

NADPH Rat Liver Microsomal Metabolites Study for TC-I-165 Test Methods

TC-I-165 was used for a NADPH dependent rat liver microsomal metabolitesstudy, and the corresponding sulfoxide, TC-II-68, and sulfone, TC-II-70,also were prepared (FIG. 14).

Experiments were generally carried out according to the generalprocedure from the microsomal stability study of TC-I-165. After 0, 5,10, 20, 40, and 60 minutes incubation with rat liver microsome, 50 μLacetonitrile was added to quench reactions. The reaction mixture wasvortexed and incubated in an ice bath for 2 h. The samples werecentrifuged to remove the precipitated protein. The supernatantsolutions were loaded directly for HPLC analysis.

Data and Results

TC-II-68 was a metabolite from TC-I-165 after 60 minutes of NADPHdependent rat liver microsome incubation. TC-II-70 is not a metabolite.Only one new peak was observed from the microsomal residue of TC-I-165.Using the same HPLC program (25% acetonitrile, isocratic, 30 minutes),the retention time of the new peak was approximately identical to theone from TC-II-68. The retention time of TC-II-70 was well seperatedfrom that of the new peak (see FIGS. 15-20).

Rat Liver Microsomal Stability Studies of TC-II-70 Test Methods

TC-II-70 was used in a NADPH dependent rat liver microsomal stabilitystudy. The test (TC-II-70), positive control (minaprine), negativecontrol (warfarin), and internal standard (haloperidol) compounds inthis experiment are listed in FIG. 21. The experiment was carried outmostly according to the general procedure from the microsomal stabilitystudy of TC-I-165. After 0, 20, and 40 minutes incubation, 100 μLacetonitrile was added to quench reactions. The reaction mixture wasvortexed and incubated in an ice bath for 2 h. Haloperidol (50 μL, 100μM) was added to every test tube as an internal standard. The sampleswere centrifuged to remove the precipitated protein. The supernatantsolutions were loaded directly for HPLC analysis.

Results

TC-II-70 is more stable in a NADPH dependent rat liver microsomalstability study compare with its sulfide, TC-I-165 (FIGS. 22-25). Table5 shows results of microsomal stability data processing.

TABLE 5 Sample Name RR Mean % Mean ± S.E. Mina 5 μM 0 min 0.711  100 ±11.5 Mina 5 μM 40 min 0.321 45.2 ± 8.1  TC-II-70 15 μM 0 min 0.237  100± 12.6 TC-II-70 15 μM 20 min 0.261  109 ± 13.3 TC-II-70 15 μM 40 min0.201 84.8 ± 21.8 War 15 μM 0 min 0.488  100 ± 7.75 War 15 μM 60 min0.470 96.1 ± 7.66

Blood Brain Barrier Studies

Preliminary blood brain barrier (BBB) penetration experiment. An in vivoBBB penetration experiment was carried out for CMB-087229. CMB-003319was used as the internal standard due to its structural similarity toCMB-003299. CMB-087229 was formulated in 100 μL DMSO in 2 mL phosphatebuffered saline. Blood concentration of CMB-087229 (MTD of 75 mg/kd IP)1 hour following a single 1 mg IP dose was approximately 10 nM. Brainconcentration of CMB-087229 1 hour following a single 1 mg IP dose wasapproximately 120 nM. After four hours, concentration in the brain was194 μM. At 3 hours, plasma concentration measured 342 μM. At 6 hours,plasma concentration measured 347 μM. At 12 hours, plasma concentrationmeasured 248 μM. At 24 hours, plasma concentration measured 1.68 μM.CMB-087229 reaches approximately 55% of the blood level in the brain.

Brain Uptake Procedure

CMB-087229 Mice Brain and Plasma Data CMB-087229 (1 mg/kg) wasadministrated to mice (Tg6799 colony with B6/SJL background) byintraperitoneal injection in a 60% PBS and 40% dimethyl sulfoxidesolution. At 10 minutes after compound administration the mice weresacrificed. Blood was harvested and plasma was transferred to flashfreeze in liquid nitrogen after centrifugation. Mice were perfused. Thebrain was homogenized in 100% acetonitrile. CMB-003319 (0.5 μg, 1.73nmol) was added as an internal standard. The homogenates werecentrifuged at 12000×g for 12 min, and the supernatant was evaporated.The residue was reconstituted in 33.3% acetonitrile. Solid phaseextraction followed by LCMS (API 300 liquid chromatography-tandem massspectrometry system, Applied Biosystems, Foster City, Calif.; Agilent1100 series HPLC system, Agilent Technologies, Wilmington, Del.) wasused to quantify the amount of CMB-087229 in mouse brain. Solid-phaseextraction cartridges (Sep-Pak C18, Water Associates, Milford, Mass.)were washed with 1 mL acetonitrile and equilibrated with 1 mL of water.The reconstituted supernatant was then loaded, washed with 1 mL×2 of33.3% acetonitrile. CMB-087229 was then dried and submitted for LCMS.

Results

It should be noted that data obtained in March were not identical todata obtained in July. However, the brain standard curve and mice brainsamples maintain fair standard deviations and linearity, and thus, theresults from the brain studies can still be quantified. In light of thisdata, it appears that some amount of CMB-003329 does penetrate the BBB(FIG. 8-10).

TABLE 6 CMB-087229 Brain Standard Curve Brain Extract Concentration(μg/mL) Brian Area Ratio (Mean ± S.E.) 0.05 0.148 ± 0.0007 0.10 0.268 ±0.0087 0.50 1.11 ± 0.155 1.00 2.48 ± 0.084

TABLE 7 CMB-087229 Plasma Standard Curve Plasma Concentration (μg/mL)Plasma Area Ratio (Mean ± S.E.) 0.01 0.068 ± 0.00042 0.05 0.304 ±0.00436 0.10 0.637 ± 0.0337  0.50 1.58 ± 0.0604

TABLE 8 CMB-087229 Plasma Standard Curve Plasma Concentration (μg/mL)Plasma Area Ratio (Mean ± S.E.) 0.01  1.210 ± 0.0374 0.025  2.068 ±0.0316 0.05  3.554 ± 0.0775 0.10 6.429 ± 0.109 0.25 16.05 ± 0.684 0.5030.22 ± 1.351 1.0 59.67 ± 3.379 2.5 149.2 ± 1.744 5.0 297.8 ± 7.539 10591.9 ± 0.758

TABLE 9 Brain Brain/ Brain Brain Extract Brain concen- Plasma plasmaArea concentration weight tration concentration Ratio Ratio (μg/ml) (g)(μg/g) (μg/ml) July July #2 0.292 0.121 0.470 0.257 0.0904 2.84 #3 0.3840.159 0.432 0.366 0.0892 4.10 #4 0.453 0.187 0.399 0.467 0.149 3.13 #5 00 0.415 0 0 0

TABLE 10 CMB-087229 Mice Brain and Plasma Data Plasma Brainconcentration concentration (Mean ± S.E.) (Mean ± S.E.) Brain/PlasmaRatio (μg/g) (μg/ml) (Mean ± S.E.) 10 min July 0.363 ± 0.105 0.110 ±0.0342 3.36 ± 0.660

Having described some illustrative embodiments of the invention, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the invention. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction.

Use of ordinal terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: p is 1-2;

designates a single or double bond, wherein one

is a single bond and one

is a double bond; each R¹ is independently —R, —OR, —SR, —S(O)R, —SO₂R,—OSO₂R, —N(R)₂, —CN, —NO₂—NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂,—NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R,—OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or an optionally substituted 3-8membered saturated, partially unsaturated, or aryl monocyclic ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an optionally substituted 8-10 membered saturated, partiallyunsaturated, or aryl bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; or two R¹ groups are takentogether to form

 wherein: X is N or C; each R⁵ is independently —R, —OR, —SR, —S(O)R,—SO₂R, —OSO₂R, —N(R)₂, —CN, —NO₂—NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂,—NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R,—OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or a 3-8 membered saturated,partially unsaturated, or aryl monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or an 8-10membered saturated, partially unsaturated, or aryl bicyclic ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein R⁵ is optionally substituted with 1-5 R groups; each R isindependently hydrogen, halogen, optionally substituted C₁₋₂₀ aliphatic,optionally substituted C₁₋₂₀ heteroaliphatic, optionally substitutedphenyl, or optionally substituted arylalkyl, or two R on the samenitrogen are taken together to form a 5-6 membered saturated, partiallysaturated, or aryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, or two R on the same carbon or onadjacent carbons are optionally taken together to form a 3-6 memberedsaturated cycloalkyl or fused monocyclic ring containing 0-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur; n is 0-1; eachR² and R³ are independently —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —C(O)R,—C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, a suitable aminoprotecting group, or an optionally substituted 3-8 membered saturated,partially unsaturated, or aryl monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or anoptionally substituted 8-10 membered saturated, partially unsaturated,or aryl bicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R⁰ is R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R,N(R)₂, —NRC(O)R, —NRC(O)(CO)R, —NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R,—N(R)SO₂R, —N(R)SO₂OR, —C(O)R, —C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂,—OC(O)N(R)₂, or

 wherein: L is a valence bond or a bivalent saturated or partiallyunsaturated C₁₋₁₀ hydrocarbon chain, wherein 1-4 methylene units of Lare optionally and independently replaced by —O—, —S—, —C(O)—, —OC(O)—,—C(O)O—, —OC(O)O—, —S(O)—, —S(O)₂—, —OSO₂O—, —N(R)C(O)—, —C(O)NR—,—N(R)C(O)O—, —OC(O)NR—, —N(R)C(O)NR—, —N(R⁶)—, or -Cy-, and wherein L isoptionally substituted with 1-4 R groups; wherein: each -Cy- isindependently a bivalent optionally substituted saturated, partiallyunsaturated, or aromatic monocyclic or bicyclic ring selected from a6-10 membered arylene, a 5-10 membered heteroarylene having 1-4heteroatoms independently selected from oxygen, nitrogen, or sulfur, a3-8 membered carbocyclylene, or a 3-10 membered heterocyclylene having1-4 heteroatoms independently selected from oxygen, nitrogen, or sulfur;R⁶ is —R, —OR, —SR, —S(O)R, —SO₂R, —OSO₂R, —C(O)R, —C(O)OR, —OC(O)R,—OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, a suitable amino protecting group, oran optionally substituted 3-8 membered saturated, partially unsaturated,or aryl monocyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or an optionally substituted 8-10membered saturated, partially unsaturated, or aryl bicyclic ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;Ring A is a 3-8 membered saturated, partially unsaturated, or arylmonocyclic ring optionally containing 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or an 8-10 memberedsaturated, partially unsaturated, or aryl bicyclic ring optionallycontaining 0-4 heteroatoms independently selected from nitrogen, oxygen,or sulfur, and wherein Ring A is optionally substituted with moccurrences of R⁴; m is 0-5; and each R⁴ is independently —R, —OR, —SR,—CN, —S(O)R, —SO₂R, —OSO₂R, —N(R)₂, —NO₂, —NRC(O)R, —NRC(O)(CO)R,—NRC(O)N(R)₂, —NRC(O)OR, —N(R)S(O)R, —N(R)SO₂R, —N(R)SO₂OR, —C(O)R,—C(O)OR, —OC(O)R, —OC(O)OR, —C(O)N(R)₂, —OC(O)N(R)₂, or a 3-8 memberedsaturated, partially unsaturated, or aryl monocyclic ring optionallycontaining 0-4 heteroatoms independently selected from nitrogen, oxygen,or sulfur.
 2. The compound of claim 1 of either of the formulae:


3. The compound of claim 1 of either of the formulae:


4. The compound of claim 1 of the formula:


5. The compound of claim 4, wherein each occurrence of R¹, R² and R³ areindependently selected from the group consisting of hydrogen andsubstituted or unsubstituted C₁-C₆ alkyl.
 6. The compound of claim 4,wherein each occurrence of R¹, R² and R³ are independently selected fromthe group consisting of hydrogen, methyl, ethyl, propyl, and butyl. 7.The compound of claim 4, wherein at least one of R¹, R², and R³ ishydrogen.
 8. The compound of claim 4, wherein at least two of R¹, R²,and R³ are hydrogen.
 9. The compound of claim 4, wherein R¹, R², and R³are hydrogen.
 10. The compound of claim 4, wherein at least one of R¹,R², and R³ are C₁-C₆ alkyl.
 11. The compound of claim 4, wherein atleast one of R¹, R², and R³ is methyl.
 12. The compound of claim 4,wherein at least two of R¹, R², and R³ are methyl.
 13. The compound ofclaim 4, wherein R¹ is hydrogen and R² and R³ are methyl.
 14. Thecompound of claim 4, wherein at least one occurrence of R⁴ isindependently halogen.
 15. The compound of claim 4, wherein at least oneoccurrence of R⁴ is independently chlorine.
 16. The compound of claim 1of the formula:


17. The compound of claim 16, wherein R⁴ is a halogen.
 18. The compoundof claim 16, wherein R⁴ is chlorine.
 19. The compound of claim 1 of anyone of the formulae:


20. The compound of claim 1 of any one of the formulae:


21. A compound of claim 20, wherein R⁴ is chlorine.
 22. A compound ofclaim 1 of any one of the formulae:


23. The compound of claim 1 of the formula:


24. The compound of claim 23, wherein each occurrence of R¹, R² and R³are independently selected from the group consisting of hydrogen andsubstituted or unsubstituted C₁-C₆ alkyl.
 25. The compound of claim 23,wherein each occurrence of R¹, R² and R³ are independently selected fromthe group consisting of hydrogen, methyl, or phenyl.
 26. The compound ofclaim 23, wherein at least one of R¹, R², and R³ is hydrogen.
 27. Thecompound of claim 23, wherein at least two of R¹, R², and R³ arehydrogen.
 28. The compound of claim 23, wherein R¹, R², and R³ arehydrogen.
 29. The compound of claim 23, wherein at least one of R¹, R²,and R³ is methyl.
 30. The compound of claim 23, wherein at least two ofR¹, R², and R³ are methyl.
 31. The compound of claim 23, wherein R¹ ishydrogen and R² and R₃ are methyl.
 32. The compound of claim 23, whereinR³ is phenyl.
 33. The compound of claim 23, wherein at least oneoccurrence of R⁴ is independently halogen.
 34. The compound of claim 23,wherein at least one occurrence of R⁴ is independently chlorine.
 35. Thecompound of claim 23, wherein m is 1 and R⁴ is chlorine.
 36. Thecompound of claim 1 of the formula:


37. The compound of claim 36, wherein R⁴ is a halogen.
 38. The compoundof claim 36, wherein R⁴ is chlorine.
 39. The compound of claim 1 of anyone of the formulae:


40. The compound of claim 1 of any one of the formulae:


41. A compound of claim 40, wherein R⁴ is chlorine.
 42. The compound ofclaim 1 of any one of the formulae:


43. The compound of claim 1 of any one of the formulae:


44. The compound of claim 1 of the formula:


45. The compound of claim 44, wherein each occurrence of R¹, R² and R³are independently selected from the group consisting of hydrogen andsubstituted or unsubstituted C₁-C₆ alkyl.
 46. The compound of claim 44,wherein at least one of R¹, R², and R³ is hydrogen.
 47. The compound ofclaim 44, wherein at least two of R¹, R², and R³ are hydrogen.
 48. Thecompound of claim 44, wherein R¹, R², and R³ are hydrogen.
 49. Thecompound of claim 44, wherein at least one of R¹, R², and R³ is methyl.50. The compound of claim 44, wherein at least two of R¹, R², and R³ aremethyl.
 51. The compound of claim 44, wherein R¹ is hydrogen and R² andR³ are methyl.
 52. The compound of claim 44, wherein at least oneoccurrence of R⁴ is independently halogen.
 53. The compound of claim 44,wherein at least one occurrence of R⁴ is independently chlorine.
 54. Thecompound of claim 1 of the formula:


55. The compound of claim 54, wherein R⁴ is a halogen.
 56. The compoundof claim 54, wherein R⁴ is chlorine.
 57. The compound of claim 1 of anyone of the formulae:


58. The compound of claim 1 of any one of the formulae:


59. A compound of claim 58, wherein R⁴ is chlorine.
 60. The compound ofclaim 1 of any one of the formulae:


61. The compound of claim 1 of the formula:


62. The compound of claim 61, wherein each occurrence of R¹, R² and R³are independently selected from the group consisting of hydrogen andsubstituted or unsubstituted C₁-C₆ alkyl.
 63. The compound of claim 61,wherein at least one of R¹, R², and R³ is hydrogen.
 64. The compound ofclaim 61, wherein at least two of R¹, R², and R³ are hydrogen.
 65. Thecompound of claim 61, wherein R¹, R², and R³ are hydrogen.
 66. Thecompound of claim 61, wherein at least one of R¹, R², and R³ is methyl.67. The compound of claim 61, wherein at least two of R¹, R², and R³ aremethyl.
 68. The compound of claim 61, wherein R¹ is hydrogen and R² andR³ are methyl.
 69. The compound of claim 61, wherein at least oneoccurrence of R⁴ is independently halogen.
 70. The compound of claim 61,wherein at least one occurrence of R⁴ is independently methyl.
 71. Thecompound of claim 61, of the formula:


72. The compound of claim 61, of any of the formulae:


73. The compound of claim 1, of any of the formulae:


74. The compound of claim 61, wherein at least one occurrence of R⁴ isindependently chlorine.
 75. The compound of claim 1 of the formula:


76. The compound of claim 75, wherein R⁴ is a halogen.
 77. The compoundof claim 75, wherein R⁴ is chlorine.
 78. The compound of claim 1 of anyone of the formulae:


79. The compound of claim 1 of any one of the formulae:


80. A compound of claim 79, wherein R⁴ is chlorine.
 81. The compound ofclaim 1 of any one of the formulae:


82. The compound of claim 1 of the formula:


83. The compound of claim 1 of the formula:


84. The compound of claim 1 of the formula:


85. The compound of claim 1 of the formula:


86. The compound of claim 1 of the formula:


87. The compound of claim 1 of the formula:


88. The compound of claim 1 of any one of the formulae:


89. The compound of claim 1 of any one of the formulae:


90. The compound of claim 1 of any one of the formulae:


91. The compound of claim 1, of any one of the formulae:


92. The compound of claim 1 of any one of the formulae:


93. The compound of claim 1 of any one of the formulae:


94. The compound of claim 1 of the formula:


95. The compound of claim 94 of the formula:


96. The compound of claim 94, wherein at least one of R³ is hydrogen.97. The compound of claim 94, wherein at least one of R³ is methyl. 98.The compound of claim 94, wherein at least one occurrence of R⁴ isindependently halogen.
 99. The compound of claim 94, wherein at leastone occurrence of R⁴ is independently chlorine.
 100. The compound ofclaim 94, wherein m is 1 and R⁴ is chlorine.
 101. The compound of claim94 of the formula:


102. The compound of claim 101, wherein R⁴ is a halogen.
 103. Thecompound of claim 101, wherein R⁴ is chlorine.
 104. The compound ofclaim 94 of the formula:


105. The compound of claim 94 of the formula:


106. The compound of claim 94 of the formula:


107. The compound of claim 94 of the formula:


108. The compound of claim 94 of the formula:


109. The compound of claim 94 of the formula:


110. The compound of claim 94 of the formula:


111. A compound of claim 105 of the formula:


112. The compound of claim 1, of the formula:


113. The compound of claim 1, of the formula:


114. The compound of claim 1, of the formula:


115. The compound of claim 1, of the formula:


116. The compound of claim 1, of the formula:


117. The compound of claim 1, of the formula:


118. The compound of claim 115, wherein m is 1 and R⁴ is halogen. 119.The compound of claim 115, wherein m is 1 and R⁴ is chlorine.
 120. Acompound of claim 1, of any one of the formulae:


121. A compound of claim 1, of any one of the formulae:


122. A compound of claim 1, of any one of the formulae:


123. A compound of claim 1, of any one of the formulae:


124. A compound of claim 1, of any one of the formulae:


125. A compound of claim 1, of any one of the formulae:


126. The compound of claim 1, of the formula:


127. The compound of claim 94, of the formula:


128. The compound of claim 94, of the formula:


129. The compound of claim 94, of the formula:


130. The compound of claim 94, of the formula:


131. The compound of claim 94, of the formula:


132. The compound of claim 94, of the formula:


133. A compound of claim 127, of the formula:


134. A compound of claim 127, of the formula:


135. A compound of claim 127, of any one of the formulae:


136. A compound of claim 1, of any one of the formulae:


137. A compound of claim 1, of any one of the formulae:


138. A compound of claim 1, of any one of the formulae:


139. A compound of claim 1, of any one of the formulae:


140. A compound of claim 1, of any one of the formulae:


141. A compound of claim 1, of any one of the formulae:


142. A compound of claim 136 of the formula:


143. The compound of claim 1 of the formula:


144. The compound of claim 143 of the formula:


145. The compound of claim 1 of the formula:


146. A pharmaceutical composition for treating ALS comprising atherapeutically effective amount of a compound of claim 1 and apharmaceutically acceptable excipient.
 147. A pharmaceutical compositionfor treating a disease associated with abnormal protein aggregationcomprising a therapeutically effective amount of a compound of claim 1and a pharmaceutically acceptable excipient.
 148. A method of treatingamyotrophic lateral sclerosis (ALS) comprising administering atherapeutically effective amount of a compound of claim 1 to a subjectwith ALS.
 149. The method of claim 148, wherein the subject is a mammal.150. The method of claim 148, wherein the subject is a mouse.
 151. Themethod of claim 148, wherein the subject is a human.
 152. A method ofinhibiting or reversing abnormal protein aggregation comprisingadministering to a subject a therapeutically effective amount of acompound of claim 1 to inhibit or reverse abnormal protein aggregation.153. The method of claim 152, wherein the protein aggregates are G93ASOD1 protein aggregates.
 154. The method of claim 152, wherein thesubject is a mammal.
 155. The method of claim 152, wherein the subjectis a mouse.
 156. The method of claim 152, wherein the subject is ahuman.
 157. A method of inhibiting or reversing abnormal proteinaggregation comprising administering to a cell a therapeuticallyeffective amount a compound of claim 1 to inhibit or reverse abnormalprotein aggregation.
 158. A method of protecting cells from thecytotoxic effects of aggregated SOD1 comprising administering to a cellan effective amount of a compound of claim 1 to protect againstaggregated SOD1.
 159. The method of claim 158, wherein the cells arePC12 cells.
 160. The method of claim 158, wherein the cells aremammalian cells.
 161. A method of modulating proteasome functioncomprising administering to a cell an effective amount of a compound ofclaim 1 to modulate proteasome function.
 162. The method of claim 160,wherein the cells are PC12 cells.
 163. The method of claim 160, whereinthe cells are mammalian cells.